Keratin-based powders and hydrogel for pharmaceutical applications

ABSTRACT

A hydratable, highly absorbent keratin solid fiber or powder capable of absorbing a large weight excess of water may be produced by partially oxidizing hair keratin disulfide bonds to sulfonic acid residues and reacting the sulfonic acid residues with a cation. The neutralized suspension can be filtered, washed, and dried, leaving keratin solid which can be shredded into fibers and further ground into powder. Addition of water to the solid produces a hydrogel. The powder or hydrogel may be useful as an absorbent material, as a therapeutic for skin, or as an excipient. The keratin materials can be incorporated into nonwoven films. The hydrogel can be used as a biocompatible viscoelastic filler for implant applications. Another use for the absorbent keratin and keratin hydrogel is as an excipient in pharmaceutical and cosmetic applications.

I. RELATED APPLICATIONS

[0001] The present application is a divisional of U.S. patentapplication Ser. No. 09/638,643 filed Aug. 13, 2000, now U.S. Pat. No.6,544,548, which is a continuation-in-part of U.S. patent applicationSer. No. 09/587,157 filed Jun. 5, 2000, which is a continuation-in-partof U.S. patent application Ser. No. 09/528,893 filed Mar. 20, 2000,which is a continuation-in-part of U.S. patent application Ser. No.09/512,918, filed Feb. 25, 2000 which is a continuation-in-part of U.S.patent application Ser. No. 09/394,782 filed on Sep. 13, 1999.

II. FIELD OF THE INVENTION

[0002] The present invention is related generally to a keratincomposition and method for making same. Specifically, the presentinvention relates to an absorbent keratin powder or fiber. Morespecifically, the present invention includes a hydratable keratin solidwhich forms a hydrogel upon addition of water for use in variousapplications including nonwoven films, diapers, skin treatments,prosthetic devices, excipients, tissue engineering scaffolds and thelike. Active agents can be bound to the keratin excipient for controlleddrug delivery.

III. BACKGROUND OF THE INVENTION

[0003] Absorbent materials are currently used to absorb body fluids suchas urine, menses, and wound exudate. The absorbent materials are placednear the skin to serve this purpose. One class of products includesdiapers, where the absorbent material can be derived from wood pulp,cellulosic fibers, or super absorbent, synthetically produced material.Diapers commonly have an inner core designed to absorb urine and water.The core is typically formed from a superabsorbent polymer dispersed ina larger amount of less absorbent material. The absorbent materialstypically contained in the core are separated from the skin by at leastone layer of material. The absorbent materials absorb urine and canbecome saturated. It is believed that some material from the absorbentcore leaches from the wet absorbent and travels back to the skin. In thecase of chemically treated absorbent materials and films, depending onthe chemicals, the leachate may be irritating and is not believed to bebeneficial. Skin contact with urine can also occur and result inirritation. This type of irritation may exacerbate diaper rash problems.

[0004] Other products which contain absorbent materials for use next tothe skin include feminine hygiene products such as tampons and pads.These products serve to absorb menses. Another class of products usingabsorbent materials includes wound dressings, both those designed forhumans, and dressings for veterinary use for application to wounds orskin irritations or disorders in animals. For specific applications,wound dressings preferably absorb exudate from wounds while keeping thewounds relatively moist to promote healing. In some applications, a gelmay be desirable as a wound dressing, where the gel can maintain a moistwound environment, while absorbing excess exudate.

[0005] What would be desirable is an absorbent material formed from anatural product. What would be beneficial is a non-toxic product derivedfrom natural sources that would cause no concern when leachate from thematerial contacts the body or the material itself contacts the body.What would be advantageous is a material that can absorb urine and, whenwet, leach out a natural product that is beneficial with respect todiaper rash. What would be desirable is a material that can return askin healing leachate to the skin. What would be desirable is a materialthat aids wound healing. What would be desirable is a hydrogel made ofnatural products formable by adding water to a powder or fiber. Whatwould be desirable is a biocompatible carrier or excipient that could beused in the delivery of drug actives to various organs of the body. Whatwould be desirable is a biocompatible carrier to which active agents canbe bound and later released.

[0006] Tissue engineering is a rapidly growing field encompassing anumber of technologies aimed at replacing or restoring tissue and organfunction. The success of tissue-engineered implants rest on theinvention of biocompatible materials that can act as cell-scaffolds andsupport cell growth. Of benefit are tissue-engineering scaffoldsmaterials that are mitogenic or contain mitogenic factors. Suchscaffolding materials can be used for in a wide array of tissueengineering implants containing cellular components, such as, forexample, osteoblasts, chondrocytes, keratinocytes, and hepatocytes, torestore or replace bone cartilage, skin and liver tissue respectively.

IV. SUMMARY OF THE INVENTION

[0007] The present disclosure addresses at least some of thedeficiencies in the art by providing a hydratable, hydrogel-formingsolid derived from a keratinous source such as hair, fur, human hair andthe like. In the context of the present invention, the term “hydratablekeratin” and “hydratable keratin material” is a keratin or keratinmaterial that when hydrated can form a hydrogel. In the context of thepresent invention, the term “cross-linked insoluble oxidized keratin”means a network of cross-linked keratin containing sulfonic acidresidues that is effectively of infinite molecular weight in that themolecular weight is approximated by the weight of the sample. Suchnetworks may be viewed as being effectively one molecule. The status ofa sample as being a “cross linked insoluble oxidized keratin” can beverified by the addition of a solvent in which an oxidized keratinmonomer (prepared by breaking all cross-links) is soluble. A“cross-linked insoluble oxidized keratin” will swell upon addition ofsuch a solvent, but will not enter solution despite agitation, heat andprolonged incubation unless and until the cross-linked insolubleoxidized keratin undergoes degradation to form lower molecularmolecules. The cross-linked insoluble oxidized keratin preparation ofthe current invention may contain up to about 90 percent by weight oflower molecular weight polypeptides which maybe soluble, but theinsoluble character and gel forming are dependent upon network structureof the cross-linked insoluble oxidized keratin. The term “insoluble”refers to the properties of the of cross-linked oxidized keratin whileit exists as a network of effectively infinite molecular weight. Uponhydrolysis, the cross-linked insoluble oxidized keratin network willenter into solution to the extent of degradation to lower molecularweight polypeptides. As this process ensues, molecules of finitemolecular weight are generated which continue to hydrolyze and degradeto molecules of decreasing molecular weight. The solubility of theselower molecular weight polypeptides is enhanced by the fact thathydrolysis results in the generation of molecules that are more solubleby virtue of their low molecular weight (Flory, 1953). Solubility isfurther enhanced by the increasing relative molar percentage of amineand acid functional groups that are being generated as a result of thecontinued hydrolytic degradation. The rate of such degradation can becontrolled by varying preparation parameters of the “cross-linkedinsoluble oxidized keratin” such as extent of oxidation and the solidscontent. When the solvent is water, a cross-linked insoluble oxidizedkeratin that has undergone the ion exchange process of the currentinvention will form a hydrogel.

[0008] In certain embodiments, a hydrogel-forming solid as disclosedherein may absorb up to 5 to 20 times its weight in water to form ahydrogel. Such a solid, as well as the hydrogel formed from the solidwill be useful in various applications such as use as an absorbent withskin healing properties when incorporated into diapers, feminine hygieneproducts, wound dressings, including both human and veterinary uses, asa soft tissue augmentation medium when used in subdermal implants,tissue engineering cell scaffolds, as a moisture containing agent incosmetics, oils, lotions, or gels for use on the skin, in applicationsrelated to the healing of damaged skin, and as a pharmaceuticalexcipient for sustained and/or controlled release pharmaceuticalapplications.

[0009] A hydratable keratin solid may be made by methods that includeproviding a keratinous material, or keratin, having disulfide linkagesand partially or substantially oxidizing the keratinous material with anoxidizing agent, for example, such that some disulfide linkages arecleaved and oxidized, forming hydrophilic sulfonic acid or cysteic acidresidues. A preferred source of keratinous material is human hair,although the keratin may be obtained from hair or fur of animalsincluding any mammal, or from finger or toenail material or from hooves,feet, beaks, skin, feather or horns. Human hair is a preferred source ofkeratin because of its ready availability from cuttings of barber andbeauty shops, because it is expected to be less prone to causeundesirable immune or allergic reactions in a human, and because akeratin preparation may be made from the hair of a subject for whom thepreparation will be used. This last advantage can be especiallyimportant in embodiments involving subdermal and tissue engineeringimplantations.

[0010] It is well known in the art that keratins contain substantialsulfur, that is, the amino acid sequence of keratin contains a highproportion of cysteine residues as compared to proteins in general.These cysteines each include a sulfhydryl moiety that is able to bondwith another sulfhydryl moiety from another cysteine residue to form adisulfide bond. The second cysteine may reside within the same keratinmolecule, or in another keratin molecule. These disulfide bonds areresponsible for much of the tertiary and/or quaternary structure of thisclass of proteins. A suitable oxidizing agent is able to break thisdisulfide bond and to oxidize one or both of the sulfhydryl moieties sothat they are no longer able to form a disulfide. Such an oxidation is apart of the process of forming the keratin products of the presentdisclosure. Preferred oxidizing agents include, but are not limited toperacetic acid, hydrogen peroxide, perborates, percarbonates, benzoylperoxide, or ammonium sulfate peroxide. However, any suitable oxidizingagent known in the art can be used in the practice of the invention.After oxidation, the liquid oxidizing agent can be filtered from theoxidized keratin solid, and the solid may be washed to remove residualoxidizing agent, for example.

[0011] The resulting solid may then be suspended in a non-aqueoussolvent and the pH may be adjusted upward with base—conveniently to atleast neutral pH. Preferred solvents for this second solution do notinclude more than about 20 volume percent water, as the water mayhydrolyze the peptide backbone during processing. Preferred solventswould include alcohols such as methanol, ethanol, or propanol, forexample, and would also include non-aqueous polar, water-misciblesolvents such as acetone and tetrahydrofuran, for example. An effectivesolvent should be able to solvate a Lewis base and should also be ableto provide a medium able to keep the keratin sufficiently swelled toallow ionic associations or interactions between the base cations andanionic sulfonic acid groups in the keratin. Small amounts of water willassist in this regard, so blends of the aforementioned solvents incombination with water up to 20 volume percent may be used. Preferredbases include, but are not limited to sodium hydroxide, potassiumhydroxide and ammonium hydroxide, which, as is known in the art, wouldyield or produce sodium, potassium and ammonium cations, respectively,upon entering solution.

[0012] The keratin suspension may be heated, and is preferably heated toboiling for a time sufficient to swell the keratin. The keratinsuspension may be stirred without heat for a longer period of time toallow a more complete association or reaction between the sulfonic acidgroups and the base cations. The continued reaction time at or near roomtemperature, or even below room temperature while stirring iscontemplated by the inventors to allow the base cations to approach andbind to the keratin anionic sites with a lower incidence of peptidebackbone degradation that could occur with continued boiling. Thecations for use in the present invention, therefore, must be able tointeract with the anionic cysteic groups in the keratin material. Theuse of the term “cations” or “monovalent cations” in the presentdisclosure and claims is indication of those cations that are able to doso. Salts of aspartate and glutamate may also be present in highconcentration and will contribute to the absorbency of the hydratablekeratin material. After a sufficient reaction time, the keratin solidmay be removed from the suspension by filtration, for example, anddried, leaving a solid salt formed of the keratin sulfonic acid orcysteic acid groups and base cations. This solid may be shredded into afibrous form and/or ground into a finely divided powder. This solid maybe used in certain embodiments, or it may be hydrated by adding water,for example, and the hydrogel, or viscoelastic hydrogel thus formed maybe used in certain embodiments.

[0013] In certain embodiments, an absorbent keratin layer may beincorporated into various absorbent articles such as a disposablediaper, a wound dressing, or feminine hygiene product, by adsorbing orcoating a keratin solid or hydrogel onto a layer of the article, byimpregnating a component of such an article, or by associating a keratinmaterial with a nonwoven layer of such an article. In certainembodiments an absorbent keratin powder may be applied directly to skinto absorb moisture and inhibit rashes or chafing, such as diaper rash,for example. A hydratable keratin solid of the invention may have anabsorbency of 1, 5, 10, 15 or even up to 20 times its weight in water.The absorbency may be adjusted by, for example, varying the degree ofoxidation of the keratin in the process. It may thus provide asubstitute or a supplement for products such as talc and cornstarch. Theinventors have demonstrated, for example, that a fibrous or powderedform of solid keratin material as described herein can absorb about 10times its weight in water in about 10 seconds.

[0014] The hydratable keratin solids as described herein form a hydrogelor a viscoelastic hydrogel upon application of water, and also arecontemplated to contain skin healing peptides associated with thekeratin, which may leach out of the keratin products when wet. Thekeratin products thus provide an added benefit, in addition to waterabsorbency, that is, healing or soothing peptides are also released thatmay have beneficial effects on the skin of a user of the products. Thisproperty offers certain benefits in embodiments such as wound dressings,as well as cosmetics, gels or lotions for application to the skin.

[0015] In certain embodiments a keratin absorbent as disclosed hereinmay be used as a wound dressing material to absorb wound exudate bydirect application, or by incorporation into a dressing. The solid,hydratable forms of keratin offer certain advantages in suchapplications because they may be stored as dry powders or fibers andhydrated to form a gel in the field, or only as needed, for example.Medical applications, such as wound exudate management or drug release,can make use of the keratin material in absorbent powder, fiber, wovenfiber, or felt form.

[0016] The keratin hydrogel is also believed to be suitable for use asan implant filler, for example, used to fill a breast implant, or toaugment soft tissue for cosmetic, reconstructive or aesthetic reasons,or in a tissue expander application. The keratin product may also beused in cosmetics to retain moisture next to the skin. The performanceof cosmetics which reduce the greasy appearance of skin can be enhancedthrough the use of moisture absorbent keratin material as an additive orbase ingredient, for example, in a cosmetic formulation. The keratinabsorbent and hydrogel can also be used for a variety of tissueengineering applications. Both materials may act as biocompatiblescaffolds that provide a mitogen, the keratin peptide, to the cellularcomponents of a tissue-engineered implant. In the case of a keratinhydrogel tissue engineered implant, the degradation of keratin to lowermolecular weight peptides can be controlled through a combination ofprocessing and formulation parameters. As with other materials known inthe art, the degradation rate is directly related to the rate ofresorbtion in-vivo (Agrawal, 1997). Therefore, the resorbtion rate ofthe keratin hydrogel can be directly controlled.

[0017] The present invention may be described, therefore, in certainaspects as a composition comprising a hydratable keratin solid, whereinthe solid comprises a keratin where at least a portion of the cysteicgroups of the keratin are ionically or electrostatically associatedwith, or may be ionically bound to cations. As used herein, ionicallybound or ionically associated would have their ordinary meaning as isknown in the art, and would include the electrostatic attraction betweenan anion and a cation, and would include such interactions directly,such as through formation of ionic bonds, and interactions throughintermediary bipolar moieties, for example. A cysteic group wouldinclude cysteine and derivatives of cysteine including cysteine andcysteic acid or sulfonic acid. As used herein, cysteic acid and sulfonicacid denote a cysteine side chain in which the terminal sulfur is bondedto three oxygen atoms to produce the sulfonic acid ion, SO₃ ⁻, or theacidic form, SO₃H. In certain embodiments, a portion of the cysteicgroups are oxidized to sulfonic acid or cysteic acid groups. Sulfonicacid or cysteic acid groups may comprise a significant portion of thetotal cysteic groups and in some embodiments the sulfonic acid groupsmay constitute a major portion of the total cysteic groups. The extentof the oxidation may be adjusted by adjusting certain parameters of theoxidation reactions, such as temperature, concentration of oxidizingagent, and time of reaction, for example, to achieve a product withcertain desired properties, such as absorbency or resiliency, forexample.

[0018] In certain embodiments, therefore, the present invention may bedescribed as a hydratable keratin solid made by a process comprisingoxidizing a portion of the cysteic acid groups of a keratin to obtain akeratin having oxidized cysteic groups, and contacting the keratinhaving oxidized cysteic groups with monovalent cations under conditionseffective to form ionic associations or ionic bonds between at least aportion of the oxidized cysteic groups and the cations.

[0019] In some embodiments, the hydratable keratin solid is made by aprocess comprising oxidizing at least a portion of the cysteic acidgroups of a keratin to obtain a keratin having oxidized cysteic groups,and contacting said keratin having oxidized cysteic groups withmonovalent cations under conditions effective to form ionic associationsor ionic bonds between a substantial portion of said oxidized cysteicgroups and said cations. The oxidation may comprise placing the keratinin a solution containing a concentration of an oxidizing agent effectiveto oxidize a portion of the cysteic acid groups. The portion of oxidizedcysteic groups may be a major portion of the total cysteic acid groups.

[0020] In certain embodiments of the present invention, the oxidationcomprises placing the keratin in a solution containing a concentrationof hydrogen peroxide, peracetic acid, perborates, percarbonates, benzoylperoxide or ammonium sulfate peroxide effective to oxidize a portion ofthe cysteic groups.

[0021] The process of the present invention may further comprise heatingthe keratin solid containing oxidized cysteic groups in a solventsolution containing a dissolved base, wherein the base produces themonovalent cations in the solution. The solvent solution may comprise asolvent selected from methanol, ethanol, propanol, ether,tetrahydrofuran (THF), acetone, propylene glycol, 1,4-dioxane, andglycol ether, or combinations of these with up to 20 volume percentwater. In certain embodiments the process further comprises removing thesolution from the heat and stirring for a time effective to form ionicbonds between the cysteic groups and cations produced by the base. Theprocess may also further comprise drying the keratin solid, such as bydrying a solid or solution under vacuum.

[0022] Another aspect of the present invention is a compositioncomprising a keratin hydrogel wherein the hydrogel is produced by addingwater to a composition comprising a hydratable keratin solid, whereinthe solid comprises a keratin where at least a portion of the cysteicgroups of the keratin are ionically bound to cations. In someembodiments, the composition of the present invention comprises akeratin viscoelastic hydrogel produced by adding water to a compositioncomprising a hydratable keratin solid, wherein the solid comprises akeratin where a portion of the cysteic groups of the keratin areionically bound to or associated with cations.

[0023] Another aspect of the present invention is a process for making ahydratable keratin solid comprising: (1) oxidizing keratin in a firstsolution comprising a soluble oxidizing agent, such that a portion ofthe disulfide bonds of the keratin are oxidized to form sulfonic acidresidues, to obtain an oxidized solid fraction; (2) separating theoxidized solid fraction from the first solution; (3) contacting theoxidized solid fraction with a second, basic solution comprising amonovalent cation dissolved in a solvent or solvent mixture; (4)maintaining the second solution containing the oxidized solid fractionand the monovalent cations for a time and at a temperature effective toallow an interaction between the sulfonic acid residues and themonovalent cations to obtain a salt solution of the keratin and themonovalent cation; and (5) substantially removing the solvent from thesalt solution to obtain a pure hydratable keratin solid.

[0024] The process may also further comprise adjusting the pH of thesecond solution, to obtain a substantially neutral solution. In someembodiments, the keratin is obtained from hair, fur, skin, feet, beaks,horns, hooves or feathers and is preferably obtained from human hair.

[0025] In some embodiments, the keratin is oxidized by suspending thekeratin in a solution of a suitable oxidizing agent such as one selectedfrom the group consisting of hydrogen peroxide, peracetic acid,perborates, percarbonates, benzoyl peroxide, and ammonium sulfateperoxide, in a concentration of between about 1 and about 35weight/volume percent. In various embodiments, the keratin is oxidizedby suspending the keratin in a solution of an oxidizing agent selectedfrom the group consisting of hydrogen peroxide, peracetic acid,perborates, percarbonates, benzoyl peroxide, and ammonium sulfateperoxide, in a concentration of about 1, or about 2, or about 3, orabout 4, or about 10, or about 15, or about 20, or about 30, or about32, or about 35 weight/volume percent. As used herein the termweight/volume percent refers to a solution in which the concentration isdetermined in weight percent, that is then diluted into a particularvolume, arriving at a weight/volume percent. For example, in order toarrive at the oxidant solutions described herein a “stock solution” atfairly high concentration is diluted in water. As an example, hydrogenperoxide may be purchased as a 30 weight % solution (30 grams ofperoxide per 100 grams of solution). To make 1 liter of a 2% solution ofthis, one would dilute 66.7 mL of the 30 weight % stock solution in933.3 mL of water. The net effect is to cut the stock solution 15-fold(from 30 down to 2%). This ratio is a weight to volume ratio, so theresulting solution is described as 2 weight/volume %.

[0026] In some embodiments, the keratin is oxidized by suspending thekeratin in a solution of a suitable oxidizing agent, such as oneselected from the group consisting of hydrogen peroxide, peracetic acid,perborates, percarbonates, benzoyl peroxide, and ammonium sulfateperoxide, in a concentration of between about 1 and about 35weight/volume percent, at a temperature between about 0° C. and about100° C. In other embodiments the temperature is between about 4° C. andabout 90° C., or between about 20° C. and about 100° C., or betweenabout 80° C. and about 100° C. In other embodiments, the temperature isabout 4° C., or about 90° C., or about 100° C.

[0027] The present invention may also include the process wherein thekeratin is oxidized by suspending said keratin in a solution of anoxidizing agent selected from the group consisting of hydrogen peroxide,peracetic acid, perborates, percarbonates, benzoyl peroxide, andammonium sulfate peroxide, in a concentration of between about 1 andabout 35 weight/volume percent, at a temperature between about 0° C. andabout 100° C. for a period of between 0.5 and about 24 hours, or in aconcentration of oxidizing agent of between about 1 and about 35weight/volume percent, at a temperature between about 0° C. and about100° C. for a period of between 1 and about 2 hours, or for betweenabout 2 and about 4 hours, or for between about 1 and about 4 hours, orfor a period of about 10 hours.

[0028] More specifically, the present invention may include oxidizingthe keratin by suspending the keratin in a solution of between about 1percent to about 32 percent peracetic acid at a temperature betweenabout 0° C. and about 100° C. for between about 0.5 and about 24 hours,or by suspending the keratin in a solution of about 1 percent peraceticacid at a temperature between about 0° C. and about 100° C. for betweenabout 0.5 and about 24 hours, or by suspending the keratin in a solutionof between about 4 percent peracetic acid at a temperature of about 4°C. for 24 hours, or by suspending the keratin in a solution of about 4percent peracetic acid at room temperature for about 24 hours, or bysuspending the keratin in a solution of about 4 percent peracetic acidat about 90° C. for about 10 hours, or by suspending the keratin in asolution of about 4 percent peracetic acid at a temperature betweenabout 20° C. and about 100° C. for between about 1 and about 4 hours, orby suspending the keratin in a solution of about 4 percent peraceticacid at a temperature between about 80° C. and about 100° C. for betweenabout 1 and about 2 hours, or even by suspending the keratin in asolution of about 2 percent peracetic acid at a temperature betweenabout 0° C. and about 100° C. for about 2 hours.

[0029] A second solution in the process of making the disclosedcompositions, wherein the second solution contains the oxidized solidfraction and monovalent cations may be heated, and may also be boiledfor between about 0.5 hours and about 12 hours, for between about 0.5hours and about 3 hours, or for about 1 hour. Once said solution isboiled, the solution may be allowed to continue reacting while beingstirred after removal of the heat. Alternatively, the solution may bestirred and allowed to react without the application of heat, or ofboiling temperatures. In certain embodiments, the solution is allowed toreact at a temperature of between about 15° C. and about 30° C. for aperiod of between about 1 and about 24 hours, or at a temperature ofbetween about 20° C. and about 25° C. for a period of between about 1and about 5 hours, or at room temperature for a period of about 5 hours.In certain preferred embodiments the solution is heated to the boilingpoint of the solvent and boiled for 2 hours.

[0030] Certain processes as described herein are effective to produce ahydratable keratin solid, and it is an embodiment of the presentinvention that those solids may be hydrated by the addition of water toobtain keratin hydrogels, or even viscoelastic keratin hydrogels. Theterms hydrogel and viscoelastic hydrogel, as used herein, are meant tohave the art recognized definition, and could be described as absorbingwater such that the water cannot be removed by mechanical methods suchas pressure or centrifugation. Viscoelastic hydrogels would also bedefined as gels that display non-Newtonian fluid properties.

[0031] In certain embodiments the present invention may be described asa disposable diaper that includes a hydratable keratin solid, or adiaper which incorporates a hydratable or absorbent keratin solid. Ahydratable keratin solid may be coated on a layer of the diaper, eithera layer next to the skin of a wearer, or a layer separated from the skinof a wearer by a water permeable layer. In certain embodiments ahydratable keratin solid may be associated with a nonwoven layer of adiaper, or may be impregnated into a layer of a disposable diaper, or itmay be contained in an inner absorbent core.

[0032] In certain alternative embodiments, the present invention may bedescribed as a feminine hygiene product, or a wound dressing thatincludes a hydratable keratin solid. As was described for use indiapers, a hydratable keratin may be coated on a layer of a product,associated with a nonwoven layer of a product, or even impregnated intoa layer of a product or contained in an absorbent core. Exemplaryproducts would include wound dressings, tampons, and sanitary pads.Wound dressings include absorbent wound dressing, that is dressingcapable of, but not limited to, absorbing wound exudate and blood.Absorbent dressings include, but are not limited to, adhesive bandages.Adhesive bandages typically comprise an absorbent pad, a backing and apressure sensitive adhesive to maintain the dressing in place. In oneaspect of the present invention, an absorbent wound dressing comprisinghydratable keratin is an absorbent pad of an adhesive bandage. In oneembodiment, the hydratable keratin in such an absorbent pad is in anonwoven film.

[0033] Certain embodiments of the invention may be described as methodsfor promoting healing of skin in a subject including a human or ananimal having damaged skin, including providing an absorbent, keratinmaterial, wherein a portion and preferably a substantial or majorportion of the cysteic groups of said keratin are oxidized and whereinwater soluble peptides are associated with the keratin, wherein at leastsome of said peptides can leach out from said keratin upon applicationof water, and wherein said peptides promote healing of damaged skin; anddisposing the absorbent keratin material near damaged skin, such thatmoisture causes at least some of said peptides to leach out of saidkeratin and to contact said skin. The method may be practiced withanimal or human subjects, such that either animal or human skin ishealed by this method. The practice of the method for promoting skinhealing as described herein may include the treatment of damaged skinincluding, but not limited to diaper rash, burn, sunburn, cut, abrasion,puncture, a sore, bed sore, ulcer, diabetic ulcer, irritated skin,surgical incision, skin graft donor site, or wrinkled skin. The keratinmaterial may be incorporated in a nonwoven film. The nonwoven film maycomprise synthetic polymer webs and may also comprise natural materialssuch as cotton. It is understood that in the practice of suchembodiments, the wound of the subject being treated may exude or excretemoisture and that the absorption of such moisture by said keratin maycause the release of water soluble peptides from keratin products of thepresent invention.

[0034] In certain embodiments the present invention may be described asa method for promoting skin healing, in particular in those embodimentsin which a keratin solid or hydrogel as described herein, such as akeratin solid or hydrogel in which the keratin is obtained from humanhair, for example, is contained in, or forms a portion of a cream,lotion, or gel for application to skin, hair, lips, or nails, forexample. Such formulations can offer various advantages such asmoisturizing the skin, or inhibiting loss of moisture from the skin, aswell as providing the healing effects of peptides that may leach fromthe keratin containing product. Such creams, lotions and gels may beapplied to damaged skin, such as dry, burned, sunburned, wrinkled, cut,scraped, chapped, irritated, ulcerated or otherwise damaged skin orother tissue.

[0035] One aspect of the present invention is a nonwoven filmcomposition comprising a synthetic polymer and a keratin material,wherein the keratin material has been oxidized and contains sulfonicacid groups. The keratin material may be oxidized keratin material thathas not undergone the ion exchange process or maybe oxidized keratinthat has undergone the ion exchange process to form a hydratablekeratin. In the latter, the sulfonic acid groups of the hydratablekeratin are associated with monovalent cations. The keratin materialsmay also be associated with pharmaceutical agents which may be in theform of polar compounds which are capable of binding or otherwiseassociating with the keratin. Such a pharmaceutical agent is asprin. Thesynthetic polymer may be, but is not limited to, α-olefins, acrylates,urethanes, acetates, nylons, esters, and copolymers thereof. An α-olefinis considered to be any monomer containing an α-double bond. Thenonwoven composition may also further comprise a natural material whichmay be, but is not limited to, cotton. In some embodiments of theinvention, the nonwoven composition is a laminate, which may be, but isnot limited to, a tri-laminate comprising two outer layers of syntheticpolymer and a middle layer of keratin material. The keratin material inthe middle layer may be partially exposed by openings in the two outernonwoven synthetic polymer layers. In some embodiments of the inventionthe synthetic polymer layers are nonwoven webs of polymer fibers. Inother embodiments, the synthetic polymer layers are woven webs ofpolymer fibers.

[0036] Another aspect of the invention is a nonwoven tri-laminatecomposition comprising a middle layer of a keratin material between twoouter layers of synthetic polymer material. The synthetic polymer may bein the form of a nonwoven web. The keratin material may be oxidizedkeratin that contains sulfonic acid residues. The oxidized keratin maybe subjected to ion exchange such that the keratin material is ahydratable keratin material. The keratin material may be associated withpharmaceutical agents, which may be in a cationic form. The syntheticpolymer may be, but is not limited to, α-olefins, acrylates, urethanes,acetates, nylons, esters, and copolymers thereof. The synthetic polymermay be, but is not limited to, poly(hydroxy acids) such as polylacticacid, polyglycolic acid, or a copolymer thereof. The nonwovencomposition may also further comprise a natural material which may be,but is not limited to cotton.

[0037] One aspect of the invention is a process for making a nonwovenfilm. In one embodiment a keratin material is applied to a firstnonwoven web layer of synthetic polymer. A second nonwoven web layer ofsynthetic polymer is applied over the keratin material so as to form atri-laminate composition with two outer layers of nonwoven syntheticpolymer web and a middle layer of keratin material. Another aspect ofthe invention is a product made by the above described process. Thekeratin material may be oxidized keratin that contains sulfonic acidresidues. The oxidized keratin may be subjected to ion exchange suchthat the keratin material is a hydratable keratin material. The keratinmaterial may be associated with pharmaceutical agents, which may be in acationic form. The synthetic polymer may be, but is not limited to,α-olefins, acrylates, urethanes, acetates, nylons, esters, andcopolymers thereof. The nonwoven composition may also further comprise anatural material which may be cotton. The keratin material in the middlelayer may be partially exposed by openings in the two outer nonwovensynthetic polymer layers.

[0038] Other aspects of the present invention include wound dressings,diapers and feminine hygiene products which comprise a nonwoven filmmade from a synthetic polymer and a hydratable keratin material. Incertain embodiments, the non-woven film of the present invention may benext to the skin or other epithelial layer of a subject, or may beseparated from the skin or other epithelial layer of a subject by awater permeable layer, which may be a non-wetting water permeable layer.In certain embodiments a hydratable keratin solid may be associated witha nonwoven layer of a diaper, or may be impregnated into a layer of adisposable diaper, or it may be contained in an inner absorbent core.These products may be laminate compositions, which may be tri-laminatescomprising two outer layers of synthetic polymer and a middle layer ofkeratin material. The keratin material in the middle layer may bepartially exposed by openings in the two outer nonwoven syntheticpolymer layers. In some embodiments of the invention the syntheticpolymer layers are nonwoven webs of polymer.

[0039] Creams, lotions, or gels of the present invention may incorporateor replace other ingredients known in the art, including, but notlimited to oleaginous, emulsifiable, emulsion base, or water-solubleointment bases as are well known in the pharmaceutical arts. Oleaginousbases that may be combined with the keratin compositions includeointments containing white wax and/or white petrolatum, ointmentscontaining yellow wax and petrolatum, cetyl esters wax, oleic acids, andparaffins. Absorbent ointment bases or emulsifiable bases that may beused include those containing anhydrous lanolin, or combinations ofcholesterol, stearyl alcohol, white wax and petrolatum, for example.Emulsion bases and components that may be used include ointmentscontaining cetyl alcohol, and cold creams such as those containing cetylesters wax, white wax, mineral oil, sodium borate and water, forexample. Other ointments of the present invention may contain glycerylmonostearate, lanolin, stearic acid, or a combination of methylparaben,propylparaben, sodium lauryl sulfate, propylene glycol, stearyl alcoholand white petrolatum, for example, or an ointment containing cetylesters wax, white wax, almond oil, sodium borate, stronger rose water,and rose oil, for example. Water soluble ointments and creams for use inthe present invention may include glycol ethers and derivatives thereof,polyethylene glycols, polyoxyl 40 stearate, and/or polysorbates.

[0040] The preparations as described herein for topical applications mayalso include protectives and absorbents, demulcents such as benzoin,hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinylalcohol, propylene glycols, sodium alginates, and tragacanth.Emollients, astringents, or antiperspirants may also be included in thekeratin containing formulations as described herein.

[0041] An aspect of the present disclosure is a method for augmentingsoft tissue in a subject comprising injecting a keratin composition asdescribed herein subdermally in an area in need of augmentation. Avariety of such applications are available in light of the presentdisclosure and would include augmentation of soft tissue including, butnot limited to bulking of a urinary sphincter in order to alleviateurinary incontinence, augmentation of vocal chords to restoreelasticity, as well as improvement of the appearance of a subject byaugmentation of breasts, lips, chin, gluteal area, or even to improvewrinkled or acne scarred skin, or skin scarred by other conditions, andincluding soft tissue voids or indentations. A keratin composition maybe provided as a dry solid and hydrated after subdermal implantation, ora hydrogel or viscoelastic hydrogel maybe prepared and implanted. Incertain embodiments, a dry or hydrated keratin material may be containedin a biocompatible envelope, bag, or container for subdermalimplantation, and hydrated after implantation by addition of water orabsorption of body fluids, or a keratin material may be suspended in aninjectable carrier and injected in the desired area of augmentation. Inone embodiment, soft tissue augmentation is accomplished by injecting apreparation comprising a keratin hydrogel and a cell population.

[0042] Another aspect of the present invention is the use of keratincompositions for tissue engineering applications. One embodiment is animplantable preparation comprising a keratin hydrogel and a cellpopulation. The cell population may include, but are not limited to,keratinocytes, fibroblasts, chondrocytes, hepatocytes, splenocytes,neurocytes, osteoblasts, or endothelial cells. The keratin hydrogelpreparation may be prepared such that it is implantable by injection.Through processing and/or formulation parameters, the keratin hydrogelmaybe prepared such that is resorbable and that such resorption is at acontrolled rate. The keratin hydrogel may be prepared such that it isresorbed upon implantation after about 150 days, or about 100 days, orabout 90 days, or about 80 days, or about 70 days, or about 60 days, orabout 50 days, or about 40 days, or about 30 days, or about 20 days, orabout 10 days, or between about 10 and about 90 days, or between about20 and about 90 days, or between about 50 and about 90 days, or greaterthan 90 days. The keratin hydrogel may also contain a therapeutic agent,which may be a water soluble peptide, which may be a mitogen.

[0043] Another embodiment is a cell scaffold comprising a nonwoven filmwhich comprises a hydratable keratin which contains sulfonate groups.The nonwoven film may comprise a synthetic polymer which may beresorbable. The hydratable keratin may also be made resorbable, therebyrendering the entire nonwoven film resorbable. The nonwoven film mayalso be a laminate composition. The hydratable keratin may contain atherapeutic agent which may be a water soluble peptide, which may be amitogen.

[0044] Another embodiment of the present invention is a method ofimplanting a preparation comprising a population of cells and a keratinhydrogel into an animal. The cell populations may include, but are notlimited to, keratinocytes, fibroblasts, chondrocytes, hepatocytes,splenocytes, neurocytes, osteoblasts, or endothelial cells. The hydrogelmay contain a therapeutic agent which may be a water soluble peptide,which may be a mitogen. A further embodiment is a method of implanting acell scaffold comprising a nonwoven film that comprises a hydratablekeratin which contains sulfonate groups. Cells may be grown on thenonwoven cell scaffold in vitro. Thereby, the nonwoven cell scaffold maybe seeded with cells prior to implantation. Such cells include, but arenot limited to, keratinocytes, fibroblasts, chondrocytes, hepatocytes,splenocytes, neurocytes, osteoblasts, or endothelial cells. The nonwovencell scaffold may be used to repair damaged hard and soft tissues. Hardtissues include, but are not limited to, bone and cartilage. Softtissues include, but are not limited to, skin, mucosa and muscle.Mucosal tissue includes, but is not limited to, gingival tissue. whichmay be damaged bone or damaged cartilage. Another embodiment comprisesimplanting a keratin hydrogel cell scaffold. A therapeutic agent may beincluded in the keratin component of all the cell scaffolds of thepresent invention. Such a therapeutic agent may leach out of the keratinand may be a water soluble peptide, which may be mitogenic.

[0045] On one aspect of the present invention, the keratin compositionis a preparation comprising a cross-linked insoluble oxidized keratinexcipient. The preparation may be in the form of a powder, tablet, film,capsule, lotion, cream, gel, solution, suspension, emulsion or aerosol.The preparation may be a cosmetic preparation or may be a pharmaceuticalpreparation. The preparation may further comprise one or more additivessuch as diluents, fillers, lubricants, stabilizers, binders and gelants.

[0046] It is an aspect of the present invention that a keratincomposition as described herein, and in particular keratin obtained fromhuman hair is also useful as an excipient for the delivery of an activeagent. An embodiment of the invention maybe described, therefore, as acomposition comprising a keratin having oxidized cysteic groups and anactive agent or as a cross-linked insoluble oxidized keratin excipientwith an active agent. In certain embodiments the active agent isphysically or sterically entrapped within the keratin excipient andreleased over time by diffusion, or as a keratin excipient is degraded.Further, in some embodiments the active agent may be associated with thekeratin excipient. The association between the active agent and thekeratin excipient may be by non-covalent attraction or association,through electrostatic, hydrophilic or ionic interaction, for example, orit may be covalently attached to a keratin excipient by covalent bondingto an oxidized keratin as described herein. In one embodiment, theactive agent is in a cationic form that ionically binds to the sulfonategroups of the ionized keratin. In another embodiment the active agent isassociated with the keratin excipient by Van der Waal's forces.Association of the active agent with a keratin excipient allows for thesustained and/or controlled release of active agents. In someembodiments, the controlled release of the active agent is provided bythe hydrolysis of the keratin excipient. Such a formulation may includea hydratable keratin solid excipient, or a keratin hydrogel depending onthe particular application. In some embodiments the active agent is apharmaceutical agent while in other embodiments the active agent is acosmetic agent.

[0047] In the practice of the invention, a dry hydratable keratin asdescribed herein may be mixed with a powdered pharmaceutical agent andwater added to hydrate the mixture, or alternatively such a solidmixture may be formulated as a compressed tablet to be orallyadministered or for extemporaneous preparations for injection, or as amolded tablet, or it may be enclosed in a capsule for oraladministration or subdermal implantation, for example. In certainembodiments a solution containing a water soluble drug or pharmaceuticalagent may be added to a hydratable keratin so that the agent is carriedinto a hydrogel along with the water. A prepared hydrogel, or dryformulation may also be enclosed in a digestible or biodegradablecapsule, such as a hard gelatin capsule for oral administration. Incertain embodiments, the described pharmaceutical preparations may beformulated for injection, either intravenous, subcutaneous, orintramuscular, for example, or for inhalant, for eye, ear, or nosedrops, or for administration as a suppository.

[0048] It is understood that the pills formulated for oraladministration, including a hydratable keratin solid, or even pills,capsules or tablets containing a keratin hydrogel may containingredients to serve as coatings, additional fillers, binders and forcolor coding purposes. These ingredients are in common use in presentpharmaceutical formulations and may include, but are not limited to,gelatin, lactose, corn starch, calcium phosphate, povidone, magnesiumstearate, stearic acid, colloidal silicon dioxide, hydroxypropylmethylcellulose, polyethylene glycol and one or more of the followingdyes: FD&C Blue No. I Lake, FD&C Blue No.2 Aluminum Lake, D&C Green No.5, D&C Yellow No. 10, FD&C Yellow No. 6 or FD&C Red No. 3. Of coursethese are only exemplary fillers and dyes, those of skill in the artwill recognize that other inactive ingredients may be used in thepreparation of the formulations of the present invention.

[0049] Keratin excipient preparations as described herein may beprepared for oral administration, and would also include injectablesolutions or suspensions for intramuscular or subcutaneous implantationincluding long acting injections, suppositories, topical ointments,transdermal applications such as skin patches, and preparationsdelivered by inhalation. Other ingredients may include a surfactant,such as hydroxypropylcellulose. Dispersions can also be prepared inglycerol, liquid polyethylene glycols, and mixtures thereof and in oils.Under ordinary conditions of storage and use, these preparations maycontain a preservative to prevent the growth of microorganisms.

[0050] Keratin excipient preparations may also include other compoundssuch as diluents, fillers, lubricants, stabilizers, binders and gelants.Diluents and fillers are added to increase bulk formation, andlubricants to reduce friction during the tableting or other formulationprocess. Binders are used in tableting and provide the cohesivenessnecessary for bonding together the ingredients under compression. Theyalso increase the strength of the compressed tablet and decrease itsfriability, leading to an improvement in the both appearance andmechanical characteristics.

[0051] The pharmaceutical forms suitable for injectable use includesterile aqueous solutions or dispersion, sterile powders and hydrogelsfor the extemporaneous preparation of sterile injectable solutions,dispersions or gels. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The prevention of theaction of microorganisms can be brought about by various antibacterialand antifungal agents, for example, parabens, chlorobutanol, phenol,sorbic acid, thimerosal, and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars or sodiumchloride.

[0052] Suitable pharmaceutical agents for use with the excipientsdescribed herein would include any pharmaceutical agent that can form anassociation with the keratin formulations through non-covalent,covalent, or steric; interaction. These agents would include proteintherapeutic agents, such as growth factors. In regard to oraladministration, such agents may include compounds such as acetaminophen,tetracyclines, penicillins, vitamins, antacids, non-steroidalantiinflammatory agents, anesthetics, breath fresheners, and minerals,for example.

[0053] In those embodiments in which transdermal administration isdesired, the disclosed compositions may be formulated to be administeredby use of a skin patch, or transdermal delivery system. Transdermaladministration may be accomplished by any of a number of systems knownin the art. Examples of systems that may be adapted for use with thecompositions described herein include those systems of transdermaladministration described in U.S. Pat. No. 4,816,252; U.S. Pat. No.5,122,382; U.S. Pat. No. 5,198,223; U.S. Pat. No. 5,023,084; U.S. Pat.No. 4,906,169; U.S. Pat. No. 5,145,682; U.S. Pat. No. 4,624,665; U.S.Pat. No. 4,687,481; U.S. Pat. No. 4,834,978; and U.S. Pat. No. 4,810,499(all incorporated herein by reference).

[0054] These methods typically include an adhesive matrix or drugreservoir system and may include a skin permeation enhancement agentsuch as ethanol, polyethylene glycol 200 dilaurate, isopropyl myristate,glycerol trioleate, linolenic acid saturated ethanol, glycerolmonooleate, glycerol monolaurate, n-decyl alcohol, capric acid, andcertain saturated and unsaturated fatty acids, and their esters,alcohols, monoglycerides, acetate, diethanolamides andN,N-dimethylamides (See for examples, U.S. Pat. No. 4,906,169).

[0055] The release rate of an active agent from a keratin excipientpreparation, when that active agent is not associated with the keratinexcipient, is determined by the rate at which water is absorbed and thekeratin solid disintegrates. The water absorption rate of the solidkeratin can be controlled by the number of sulfonic acid residuesgenerated in the oxidation procedure. By exposing the keratin sourcematerial to extremes of oxidant concentration, temperature, and time,extremes of absorption rate can be obtained. For example, at low oxidantconcentration, colder temperatures and short time periods, relativelyfew disulfide residues will be converted to sulfonic acid residues. Sucha keratin solid, further processed as described herein will absorbrelatively little water and disintegrate relatively slowly. Conversely,a keratin solid prepared at high oxidant concentration, at boilingtemperature for a long time period, further processed as describedherein, will absorb relatively large amounts of water and disintegraterelatively quickly. Disintegration rates between these extremes can beobtained by processing the keratin source material using intermediateconditions. When the active agent is associated, such as ionically, withthe keratin excipient, the release rate is determined by both the rateat which water is absorbed and the keratin solid disintegrates and therate of dissassociation of the active agent from the keratin excipient.For some materials which are so tightly bound that release bydissassociation alone is ineffectual, degradation of the keratin solidmust occur before the drug molecule can become dissolved in thesurrounding media. The release rate under these conditions can becontrolled by the degradation rate of the keratin solid. In general,oxidation and formulation conditions will effect the hydrolyticstability of the hydrogel containing the drug compound. Peracetic acidoxidized keratin provides a more hydrolytically stable gel than doeshydrogen peroxide oxidized keratin, for example. Parameters such asoxidant, oxidation time and solids content of the hydrogel have beenshown to be important parameters in controlling in-vitro stability atbody temperature.

[0056] In some embodiments of the invention, a composition for thedelivery of pharmaceutical agents is in the form of a nonwoven filmcomprising a synthetic polymer and a keratin material. The syntheticpolymer may be, but is not limited to, α-olefins, acrylates, urethanes,acetates, nylons, esters, and copolymers thereof. In some embodimentsthe nonwoven composition is a laminate, which may be a tri-laminatecomprising two outer layers of synthetic polymer and a middle layer ofkeratin material. The keratin material in the middle layer may bepartially exposed by openings in the two outer nonwoven syntheticpolymer layers. In some embodiments of the invention the syntheticpolymer layers are nonwoven synthetic polymer webs. The nonwoven filmpharmaceutical delivery composition may be used externally orinternally.

V. DETAILED DESCRIPTION OF THE INVENTION

[0057] The present invention includes a hydratable solid derived fromkeratin that is highly absorbent and can form a hydrogel or viscoelastichydrogel upon the application of water. The keratin solid can includeprotein having an ionizable pendant group such as sulfonic acid that canbe derived from an oxidized protein disulfide linkage. A preferredsource of protein is keratin, and particularly preferred is keratinobtained from hair, including human hair. While hair is a preferredsource of keratinous material, other keratinous materials are alsobelieved suitable for use in the present invention. Examples of othersources include animal hair, skin, hooves, feathers, beaks, feet andhorns. The patient or a human donor are some preferred sources of hair,as hair from these sources is most likely to result in a non-immunogenicproduct, although animal hair may be acceptable for many individuals formany applications. In one method according to the present invention,hair is provided, preferably clean and unbleached. In another method,the hair is washed with Versa-Clean™ (Fisher Scientific, Pittsburgh,Pa.), rinsed with deionized water, and allowed to dry.

[0058] A. Preparation of Oxidized Keratin

[0059] The hair can be oxidized in peracetic acid or another suitablereagent such as H₂O₂. One method utilizes between about 1% to 32%peracetic acid, at a temperature between about 0 degrees C. and 100degrees C. for between 0.5 and 24 hours. In one method, about 1weight/volume percent peracetic acid is used. One method treats 30 gramsof hair with 500 mL of 4% peracetic acid at 4 degrees C. for 24 hours.Another method treats the hair at room temperature for 24 hours. Yetanother method treats the hair at about 90 degrees C. for about 10hours. In a preferred method, the hair is treated by heating the hair inthe oxidizing agent for between about 1 and 4 hours at a temperaturebetween about 20 and 100 degrees C. In a more preferred method, the hairis treated by heating the hair in the oxidizing agent for between about1 and 2 hours at a temperature between about 80 and 100 degrees C. In amost preferred method, the hair is treated by heating the hair in about2 weight/volume percent oxidizing agent for about 2 hours at atemperature of about 100 degrees C. The oxidation is believed to cleavea significant portion of keratin disulfide bonds forming cysteic acidresidues having sulfonic acid groups. The sulfonic acid groups arebelieved to be hydrophilic in nature and will ionically bond to cationslater in the process, forming a salt of the keratin and cation. Thepartial oxidation is also believed by Applicants to release existingshort chain peptides, or form additional short chain peptides, which canremain associated with, or entrained in the keratin structure.

[0060] After oxidation, the keratin solid can be recovered from theoxidizing liquid using filtration or other suitable methods such ascentrifugation or decantation. The recovered, oxidized solid can bewashed with water or alcohol such as methanol or ethanol to remove theexcess oxidizing agent. In a preferred embodiment, washing is limited toavoid removing too much of any soluble peptide chains entrained in thekeratin.

[0061] B. Preparation of Hydratable Keratin

[0062] The solid fraction can be suspended in a suitable solvent orsolvent mixture. The solvent should be capable of at least suspendingthe hair or keratin solid and keeping the solid sufficiently swelled forsubsequent reaction. The solvent is preferably a non-aqueous solvent, asthe presence of water can act to hydrolyze peptide backbone bonds, whichcan result in an inferior product. The solvent should be able tosolubilize the later added base. One group of suitable solvents includesalcohols such as methanol and ethanol. Other solvents such as ether,tetrahydrofuran (THF), acetone, propylene glycol, 1,4-dioxane and glycolethers may also be suitable as solvents. Small amounts of water willassist in swelling the keratin and may therefore be added to theaforementioned solvents in an amount up to 20 volume percent. Thesolvent used is preferably volatile to promote evaporation from thefinal solid product.

[0063] The hair or keratin solvent suspension can then have the pHtitrated upward to at least about pH 7. Increasing the pH deprotonatesthe sulfonic acid groups, leaving the sulfonic acids free to exchangewith another cation. The pH can be adjusted with a base, preferablyhaving a monovalent cation. Preferred bases include sodium hydroxide andpotassium hydroxide.

[0064] The pH-adjusted keratin suspension can be heated for a time andtemperature sufficient to swell the keratin structure and promoteneutralizing of the sulfonic acid sites with the provided cation. In apreferred method, the keratin suspension is boiled between about 0.5hours and 12 hours. More preferably, the keratin suspension is boiledbetween about 0.5 hours and 3 hours. In one method, the keratinsuspension is boiled for about 1 hour. Boiling for too long a timeperiod leads to a mushy keratin which results from degradation of thepeptide backbone. A hydrated keratin product is less preferred due tothe greater difficulty of grinding the keratin.

[0065] After boiling, the keratin is preferably allowed to continue toreact with the provided base cation at lower temperature and withstirring. The lower temperature reaction preferably takes place at atemperature of between about 15 and 30 degrees C. for between about 1and 24 hours. More preferably, the lower temperature reaction takesplace at a temperature of between about 20 and 25 degrees C. for betweenabout 1 and 5 hours. In one method, the keratin suspension is allowed toreact with stirring at room temperature for about 5 hours. In certainembodiments the reaction is held at the boiling point of the solvent forabout 2 hours.

[0066] After reacting at lower temperature, the reacted solid can beseparated from the solvent using any suitable method such as filtration.The solid is preferably washed with a solvent such as the same solventused in the reaction. Washing the keratin removes some of the base,which is preferably removed. The base is preferably removed to make thekeratin solid less caustic.

[0067] After filtration and washing, the keratin can be dried by amethod such as evaporation under vacuum. In one method, the keratin isdried at room temperature under about 5 mm Hg vacuum for about 2 hours.The dried keratin is preferably somewhat brittle, which can result in abetter product after grinding. The dried keratin can be shredded intofibers and can further be ground into a powder. The dried keratin can bedirectly ground into a powder using a mortar and pestle, a ball mill, orother means of breaking down or comminuting the dried keratin intoparticles. Alternatively, the keratin can be ground or milled in thesolvent used for said neutralization step.

[0068] One resulting hydratable fiber or powder has been observed toabsorb about 10 to 13 times its own weight in water. In one test, fibershaving a length of between one quarter and one-half inch were observedto absorb an average of 1300%+/−33% of their weight in water at atemperature of 21.5 degrees C. The fiber has been observed to absorb atleast 10 times its own weight in water within about 10 seconds. Thepowder has been observed to rapidly absorb water as well.

[0069] The fibers were also tested for various toxicity parameters andwere found to be non-toxic, non-irritating, non-sensitizing, andbiocompatible as indicated in Table 1. TABLE 1 Hydratable KeratinToxicity Testing Data Test Standard Protocol Result Acute Oral ToxicityBiological Evaluation of Medical Devices - Non-toxic at 2 g/kg Part 11:Tests for Systemic Toxicity; ISO 10993-11, 1993. Acute Dermal ToxicityBiological Testing of Medical and Dental Non-toxic at 2 g/kg Materialsand Devices - Part 10: Irritation and Sensitization Tests; ISO 10993-10,1995. Acute Irritation Biological Testing of Medical and DentalNon-irritating at Materials and Devices - Part 10: Irritation 33.3 wt. %and Sensitization Tests; ISO 10993-10, 1995. Kligman SensitizationBiological Testing of Medical and Dental Non-sensitizing at Materialsand Devices - Part 10: Irritation 33.3 wt. % and Sensitization Tests;ISO 10993-10, 1995. Material Mediated Biological Evaluation of MedicalDevices - Passed Pyrogen Assay Part 11: Tests for Systemic Toxicity; ISO10993-11, 1993. Systemic Injection Biological Evaluation of MedicalDevices - Passed Part 11: Tests for Systemic Toxicity; ISO 10993-11,1993. Reverse Mutation Biological Evaluation of Medical Devices - PassedAssay (Ames Test) Part 3: Tests for Genotoxicity, Carcinogenicity, andReproductive Toxicity, ISO 10993-3, 1992.

[0070] C. Preparation of Nonwoven Films

[0071] 1. Nonwoven Film Comprising Hydratable Keratin Fibers

[0072] Hydratable keratin fibers may be incorporated into a nonwovenfilm by admixing with synthetic fibers which serve as a binder. Such anonwoven film can be formed by mixing synthetic fibers made from, butnot limited to, α-olefins, acrylates, urethanes, acetates, nylonsesters, or copolymers thereof with water-absorbent keratin fibers andheat pressing the mixture into a film of desired thickness. Thesynthetic fibers will serve as a binder for the keratin fibers, whilenot completely encapsulating them. This morphology provides mechanicalintegrity to the film, while allowing the keratin fibers to absorbwater. The hydrated fibers can also release material which has beenshown to be beneficial for repairing damaged epithelial tissue.

[0073] Nonwoven films can be prepared by preparing nonwoven webs of asynthetic polymer and then placing a layer of hydratable keratin fibersbetween two layers of the nonwoven polymer-web. For example, a nonwovenfilm was produced by first preparing a nonwoven web measuringapproximately one half inch thick by 24 inches wide using 9 denier, 38mm length polypropylene fibers. The web was made using a Rando-Webber,(Fiber Controls, Inc. Gastonia, N.C.) air laying machine operating at2000 rpm, 12 ft./minute let off speed with a feed rate of 4 ft./minute.A web of approximately 20 feet in length was coated over half of itslength on one side with keratin fibers of approximately 2-5 mm inlength. The keratin was spread on the web using a hand sifter. Theuncoated section of the web was folded back over the coated section toform a laminate of keratin between two layers of polypropylene. Thelaminate was passed through a Sigma heated roller press (BF Perkins,Rochester N.Y.). The rolls were oil heated to 160° C. and a pressure of350 pounds per linear inch was applied. The surface of the top rollerwas textured so as to impress a texture in the finished nonwoven film.The laminate was fed through the rollers at approximately 4 ft./minuteand the polypropylene softened and pressed such that a film ofapproximately 3 mm in thickness resulted. This nonwoven film was boundtogether by the polypropylene, but retained some flexibility. Thekeratin fibers were at least partially exposed such that the film wettedeasily and the keratin became gelatinous upon addition of water.

[0074] Nonwoven films can be made by other procedures. For example, if amore open nonwoven is desired, a laminate of keratin and syntheticfibers can be prepared as described above, and this laminate processedby a through air dryer. The through air dryer is capable of heating thelaminate but does not apply pressure to the film. In this process, thesynthetic fibers can be softened and bound together, thus providing astructural matrix for the keratin fibers. The result is a nonwoven webwhich retains more of its original, open morphology. Also, films madewith synthetic fibers can sometimes be stiff. The example given aboveresulted in a film resembling burlap. If a softer film is desired,alternative fibers or blends of fibers may be used to produce thenonwoven web. A blend of cotton and polypropylene, for example, wouldprovide a softer, more pliable nonwoven film. Cotton fibers canconveniently be blended into the nonwoven web during the air laying orcarding process, prior to coating with keratin fibers. Other naturalfibers such as hemp may also be used.

[0075] These nonwoven films are produced from a loose, laminatedprecursor. However, the keratin fibers are exposed to the surface in thefinal product. Although the exemplified polymeric binder is hydrophobic,the nonwoven film wets easily and readily absorbs water. Once water isapplied to the film, the keratin fibers absorb it and swell, thusforming a hydrogel which is entrained in the unswollen binder. This typeof film is of utility as a wound dressing because of the capability ofabsorbing wound exudate and forming a hydrated, gelatinous cover overthe wound site. Such a dressing provides a closed, moist environment,conducive to wound healing. Drug actives that are useful in woundhealing applications such as antibiotics, anti-inflammatory agents,analgesics and the like may also be bound to the keratin used inproducing the nonwoven films. The nonwoven film may be produced using abiocompatible synthetic binder material such as polylactic acid,polyglycolic acid, copolymers thereof, and drug loaded keratin. Such anonwoven device is useful as a biocompatible implant for controlledand/or sustained delivery of active agents. Due to the water absorbency,these nonwoven films also have utility as components of disposablediapers, feminine hygiene products as well as any other applicationwhere a nontoxic film with biocompatibility and absorbency is desiredand the healing of damaged skin or other epithelial tissue is deemedbeneficial or necessary. These films also have utility as implantmaterials for the repair of damaged hard or soft tissues, as cellscaffolds and tissue engineering applications.

[0076] 2. Nonwoven Film Comprising Oxidized Hydratable Keratin Powder

[0077] Nonwoven films can also be prepared with oxidized keratin powder.For example, a nonwoven web measuring approximately one half inch thickby 24 inches wide was prepared using 1.7 denier, 38 mm length Fortrel®polyester fibers supplied from Wellman, Inc. (Johnsonville, S.C.). Ablend of 20 wt. % low melt and 80 wt. % high melt fibers was first mixedby hand, then run through a Garnett fine opener, and finally carded.This was done prior to laying the web to provide a homogeneous sample.The web was made using a Rando-Webber (Fiber Controls, Inc, Gastonia,N.C.) air laying machine operating at 2000 rpm, 12 ft./minute let offspeed with a feed rate of 4 ft./minute.

[0078] The web was mechanically entangled using a hydrobonder fromHoneycomb Systems, Div. (Division of Valmet, Inc., Biddeford, Me.). Thisequipment consists of a screen conveyor and a manifold of high pressurewater jets. The web passes under the water jet manifold and the force ofthe water forces the fibers through the screen, thereby entangling them.The degree of entanglement can be controlled by the mesh size of thescreen conveyor. The excess water was removed using a vacuum strippermanufactured by Evac Corporation, (Spartanburg, S.C.). This processreduced the web's thickness to approximately one eighth inch andresulted in a more tightly entangled web with more structural integritythan one produced using only the air laying technique.

[0079] Two rolls of web, 20 feet in length, were prepared using thisprocess and used to make a laminate with hydratable keratin powder. Thekeratin powder was less than 300 μm in size and was prepared asdescribed for keratin fibers. The laminate was prepared by conveying thetwo webs from separate spools and spraying the powder onto the bottomweb. Powder was sprayed using a GEMA™ powder sprayer with anelectrostatic spray gun (the electrostatic feature was not used). Thegun was operated at 2 psi with a flow of 4.5 m³/hour through thereservoir and a make-up flow of 1.5 m³/hour through the gun. Thenonwoven laminate was conveyed with a take-up winder operating at 32ft./minute. The use of a tighter web allowed small keratin particles(length of less than 1 mm) to be used without significant loss. This wasespecially important during the winding and unwinding operations priorto thermal bonding. The web could also be moistened slightly prior tospraying the keratin in order to promote adhesion.

[0080] The nonwoven laminate was passed through a Sigma heated rollerpress. The rolls were oil heated to 160° C. and a pressure of 200 to 215pounds per linear inch was applied. The surface of the top roller wastextured so as to impress a texture in the finished nonwoven film. Thelaminate was fed through the rollers at approximately 15 to 17ft./minute. This procedure resulted in a nonwoven film of approximately3 mm in thickness. The surface of the film was smoother than the filmdescribed previously and the use of polyester, rather thanpolypropylene, produced a softer, more pliable film. The keratin powderwas at least partially exposed such that the film wetted easily and thekeratin became gelatinous upon addition of water.

[0081] D. Keratin Delivery Systems

[0082] Active agents can be incorporated into a hydratable keratinexcipient to form a delivery system. By “active agent” is meant acompound, the delivery of which is the object of the application of thepreparation comprising that compound and the keratin excipient of thepresent invention. Delivery of an active agent is generally desiredbecause of a beneficial and/or desired effect or attribute imparted bythe agent upon delivery. Physical classes of active agents that can beincorporated into the keratin excipient of the present inventioninclude, but are not limited to, compounds that may ion exchange withsulfonic acid groups, those compounds that may otherwise be formulatedas hydrochlorides, compounds that form an electrostatic association withthe keratin excipient, polar agents, polynucleotide agents, andpolypeptide and peptide agents. Polypeptide agents include bothrecombinant and native polypeptides. For example, insulin is apolypeptide agent that may be incorporated into the keratin excipient ofthe present invention. Polar compounds include, but are not limited to4-acetaminophenol, aspirin and beta-lactams. Compounds that mayotherwise be formulated as hydrochlorides include, but are not limitedto phenylpropanolamine and pseudoephedrine.

[0083] The active agent may be a pharmaceutical agent. By“pharmaceutically active agent” is meant any compound commonly referredto as a “drug” and its equivalents which include any physiologically orpharmacologically active substance that produces a localized or systemiceffect or effects in animals. The term “animal” includes mammals, humansand primates such as domestic, household, sport or farm animals such assheep, goats, cattle, horses and pigs, laboratory animals such as mice,rats and guinea pigs, fishes, avians, reptiles and zoo animals. Examplesof pharmaceutical agents that may be formulated as hydrochlorides, andexamples of pharmaceutical agents in general, are found in Remington:The Science and Practice of Pharmacy, (19th ed., ed. A. Gennaro) 1995,The Pharmacological Basis of Therapeutics, by Goodman and Gilman, 6thEd., 1980, published by the MacMillan Company, London and in The MerckIndex, 11th Edition, 1989, published by Merck & Co., Rahway, N.J.,herein incorporated by reference in their entirety. A non-exhaustivelist that exemplifies some of the classes and types of pharmaceuticalagents that may be used in the present invention is provided in Table 2.TABLE 2 Analgesics aspirin, acetaminophen, morphine, oxymorphone,codeine, oxycodone Antianxiety Drugs buspirone, benzodiazepine,venlafaxine Antiarrhythmics flecainide, encainide, lidocaine, digoxin,beta-blockers, procainamide Antibacterials beta-lactams,aminoglycosides, macrolides, clindamycin, tetracylin, quinolones,sulfonamides, trimethoprim- sulfamethoxazole, sulfisoxaole,sulfasalazine, Antibiotics penicillins cephalosporins,amnioglycosidases, macrolides, fluroquniolones, chloamphenicol,rifampin, vancomycin Anticonvulsants phenytoin, ethosuximide, valproate,diazepam Antifungals amphotericin B, clotrimaozole, econazole,fluconazole, flucytosine, griseofulvin, haloprogrin, ketoconazole,itraconazole, miconazole, nystatin, tolfanate, undecylenic acid,terconazole, triacetin Antihistamines alkylamines, ethanolamines,ethylenediamines, piperazines, phenothiazines, piperidinesAnti-Inflammatories betamethasone dipropionate, clobetasol propianate,amcinonide, halcinonide, fluocinolone acetonide, betamethasone alerte,flubiprofen, ibuprofen, indomethacin, ketoprofen, mefenamic, naproxen,phenylbutazone, suldinac Antivirals acyclovir, amantidine, didanosine,inosiplex, intrathecal, ribavirin, ganciclovir, triflurdine Cytotoxicsprednisolone, azathioprine, cyclophosphamide, cyclosporine, tacrolimusCytokines inteferon alpha, interferon beta, colony stimulating factors(GM-CSF, M- CSF, G-CSF), interleukins 1 through 11, tumor necrosisfactor beta Growth Factors platelet-derived growth factor, epidermalgrowth factor, fibroblast growth factor, insulin-like growth factors,transforming growth factor beta Muscle Relaxants benzodiazepines,imidazopyridine, diphenhydramine, pyrilamine Sympathomimeticsphenylpropanolomine, phenylephrine, psudoephedrine Vitamins A, B₆, B₁₂,C, D, E, folacin, thaimin, riboflavin, niacin, pantothenic acid, biotin

[0084] The active agent may be a cosmetic agent. The term “cosmetic”used in relation to a formulation or product means a formulation orproduct that qualifies as a cosmetic under the Federal Food, Drug andCosmetic Act, 21 U.S.C. § 321(i). By “cosmetic agent” is meant an agentthat is incorporated in a cosmetic formulation or product and that agentis reported or believed to impart a beneficial and/or desirable effector attribute upon application of the cosmetic. Cosmetic agents include,but are not limited to, anti-wrinkle agents, such as retinol andalpha-hydroxy acids, polypeptide and peptide agents derived from skinproteins (e.g., keratin, collagen and elastin), sunscreens, humectants,antioxidants, vitamins, tanning agents (both artificial and those thateffect melanogensis), and whitening agents. Descriptions of cosmeticagents may be found in International Cosmetic Ingredient Dictionary andHandbook, Cosmetic Toiletries and Fragrance Association, 8th ed. 2000,and A Consumers Dictionary of Cosmetic Ingredients, Ruth Winter, ThreeRivers Printers, 5^(th) ed, 1999, both herein incorporated by reference.

[0085] In some embodiments of the present invention, the keratinexcipient may be incorporated in a nonwoven film. In other embodimentsof the present invention, the keratin excipient may be incorporated intoa drug delivery device. Examples of systems that may be adapted fortransdermal use with the compositions described herein described in U.S.Pat. No. 4,816,252; U.S. Pat. No. 5,122,382; U.S. Pat. No. 5,198,223;U.S. Pat. No. 5,023,084; U.S. Pat. No. 4,906,169; U.S. Pat. No.5,145,682; U.S. Pat. No. 4,624,665; U.S. Pat. No. 4,687,481; U.S. Pat.No. 4,834,978; and U.S. Pat. No. 4,810,499 (all incorporated herein byreference). Examples of systems that may be adapted for inhalation ofthe compositions described herein are described in U.S. Pat. No.5,884,620 and U.S. Pat. No. 5,960,792, both incorporated herein byreference.

[0086] In some embodiments, the basis of drug delivery is binding anactive agent in a keratin matrix for later release by some mechanical,chemical, biochemical or cellular mechanism. Active agents can either beelectrostatically bound or physically entrained in the matrix.Electrostatic binding can occur in the form of ionic bond formation(specific, results in tightly bound active agent) and Van der Waal'sbond formation (less specific, results in a less tightly bound drug).Entraining an active agent can be most effectively accomplished byproviding a processes that incorporates intimate mixing of the agent andthe matrix, most notably, solution processing. Multiple combinations ofthese binding mechanisms are possible, depending on the structure andfunctionality of the drug molecule.

[0087] Electrostatic binding (ionic and Van der Waal's) can beaccomplished by performing an ion exchange with oxidized keratin innonaqueous media. The media needs to solubilize the drug and effectivelysuspend, and at least slightly swell the keratin. Ionic bonds will formbetween ionic species, whereas Van der Waal's bonds will form betweenionizable functional groups of opposite partial charge.

[0088] Entrainment can be accomplished by physical mixing of the activeagent and the keratin matrix. The more intimate the mixing, the morelikely to result in tightly bound drug. This approach works best forlarge molecules such as protein therapeutics because larger moleculescan entangle more effectively than smaller ones. A most effectiveapproach to promote intimate mixing is to add an active agent andkeratin matrix in suspension or solution. This can be accomplished byadding an aqueous solution of active agent to dehydrated keratinabsorbent and forming a hydrogel, by adding a keratin hydrogel todehydrated active agent, or by adding a solution of active agent to akeratin hydrogel. The resulting drug loaded hydrogel can be processedinto a dosage for in the gel state or dried and ground into a powder.

[0089] The keratin matrix needs to be of a particle size category indosage forms to be used for inhalation or intravenous (IV) injection.Dry, solid keratin loaded with an active agent as described previouslycan be processed into a powder of specific particle size using any oneof a variety of grinding techniques known to those skilled in the artsuch as grinding or milling. Classification into a specific particlesize range can be performed by sieving. For inhalation applications,particulates of different sizes will reach specific areas of therespiratory system. For example, particles that are greater than 5microns will reach the nasopharynx. Particles between about 2 and about5 microns will reach the trachea and bronchus. To reach the alveoli,particles must typically be less than about 1 micron. It has been shownthat particles of about 3 microns or larger are deposited in therespiratory tree and become encysted. Keratin particles for drugdelivery must reach the alveoli in order to be efficiently absorbed bythe bloodstream and therefore must be less than about 1 micron.

[0090] For intravenous injections applications, the drug loaded keratinparticles need to be small enough to be metabolized in the bloodstream(i.e. absorbed by cells) and also sufficiently small enough so as not tocause blockage of the small capillaries in the circulatory system. Thelimiting size in this regard is the lesser of the two, namely the meancapillary diameter (in humans, ca. 5 microns).

[0091] For both the inhalation and intravenous injection dosage forms,the particles sizes discussed must be the fully hydrated particle size.Depending on the processing parameters of the keratin used and theconditions under which the drug is incorporated into the keratin matrix,hydration capacities will vary. Considering a maximum absorption mass ofca. 20 times, the maximum particle sizes discussed previously would bereduced by ca. 20 times. Specific swelling volumes would depend on thematerials used since resulting densities will vary.

[0092] In various embodiments, the particle size is less than 0.5micron, or less than 1.0 micron, or less than 2 microns, or less than 3microns, or less than 4 microns, or less than 5 microns, or less than 10microns, or less than 20 microns, or less than 30 microns, or less than40 microns, or less than 50 microns, or less than 100 microns. In otherembodiments the particle size is between about 0.1 micron and about 1micron, or between about 0.1 micron and about 2 microns, or betweenabout 0.1 micron and about 3 microns, or between about 1 micron andabout 3 microns, or between about 1 micron and about 5 microns, orbetween about 1 micron and about 10 microns.

[0093] The active agent delivery system of the present invention offersdistinct advantages over conventional drug dosage forms. As with mostdelivery systems, sustained or controlled release allows the level of anagent to be maintained at a more consistent concentration, therebyallowing larger doses to be administered on a less frequent basis. Inthe system described here, the chemical and material properties of thekeratin determine the properties of the dosage form. For example,loading can be varied by the availability of sulfonic acid bindingsites, which can in turn be controlled by the keratin oxidation process.Further, the disintegration and breakdown of the keratin can also becontrolled by the relative amount of disulfide crosslinks remainingafter the oxidation process. Disintegration and dissolution will effectthe release kinetics of the dosage form. For longer term releaseapplications, the release rate can be controlled by the hydrolysis rateof the keratin, which in turn can be controlled by processing andformulation parameters such as oxidant type, oxidation time, and solidscontent. When incorporated into a nonwoven wound dressing, the bounddrugs can be tailored to those most beneficial to wound healing such as,for example, antibiotics, biocides, pain medications and growth factors.

[0094] Following are examples of preparation, testing and formulation ofkeratin delivery systems:

[0095] 1. Dissolution Testing

[0096] (a) Rotating Paddle Method

[0097] Dissolution tests are performed according to the standard UnitedStates Pharmacopoeia (USP) rotating paddle method. In this method, thevolume of the dissolution medium is fixed and agitation is providedunder defined conditions by the stainless steel paddle. A 1L volume ofpurified water or simulated gastric fluid (Lot. No. 76H9312,Sigma-Aldrich, St. Louis, Mo.) was used at 37° C.±0.5° C. Thetemperature of the dissolution medium was maintained at 37° C. byimmersing the dissolution flask in a water bath. A cover was used on thesystem to avoid water loss through evaporation. After the temperatureequilibrated, the samples were allowed to sink to the bottom of thevessel before rotation of the paddle. Rotation speed was kept constantat 50 rpm.

[0098] During the test, small volumes of sample were withdrawn atvarious time points, for example, every 20 minutes within the first hour(including a 0 time point) and every hour after that for 8 hours.Samples were taken from a precise point in the dissolution flask,halfway between the surface of the dissolution medium and the top of therotating paddle, and not less than 10 mm from the wall of the vessel.The volume withdrawn during sampling was replaced with an equal volumeof media, pre-warmed to 37° C. High-performance liquid chromatography(HPLC) was performed on the samples and the amount released from thedosage form calculated based on a calibration curve generated fromsolutions with known concentrations of the compound of interest.

[0099] (b) Franz Diffusion Cell

[0100] An alternative dissolution test for membranous or film topicalformulations is by use of one-chambered Franz type diffusion cells(Franz, 1978). Franz cells may be obtained from Crown Glass (SomervilleN.J.). In these cells, one side of a membrane is in contact with anaqueous solution and the other side is open to the ambient atmosphere,unless placed within a controlled atmosphere. The keratin-containingfilm or membrane is mounted in the diffusion cell such that one side isin direct contact with the aqueous receptor solution (Lee et al., 1986).The aqueous solution is continuously stirred and kept at 32° C. by meansof a water jacket.

[0101] Samples are removed from the aqueous medium by means of thesampling port at appropriate times. For example, samples may be takenevery 20 minutes within the first hour (including a zero time point) andevery hour after that for 8 hours. An additional sample may be taken at24 hours. High-performance liquid chromatography (HPLC) or othersuitable analytical quantification is performed on the samples and theamount released from the dosage form is calculated based on acalibration curve generated from solutions of known concentrations.

[0102] 2. Albumin Containing Preparations

[0103] Samples containing albumin were made in tablet and capsule forms.Tablets were prepared by co-grinding a mixture of 80/20 dry keratinabsorbent powder/bovine albumin. The keratin absorbent powder wasprepared as described in Section VI. B supra, and the albumin waspurchased from Sigma Chemical Co., St. Louis, Mo. (Lot No. 69H1257,Fraction V, 96% purity). The solid mixture was further homogenized byshaking in a closed container. Samples of 500 mg of the powder mixturewere made into tablets using a pellet press at 30,000 psi (400 mgkeratin, 100 mg albumin). The dissolution profile of these tablets wasmeasured by the rotating paddle method.

[0104] Albumin capsules were prepared by dissolving 2 g of albumin in200 g of deionized water. The albumin solution was added to 8 g ofabsorbent keratin powder to form a hydrogel. The hydrogel was thoroughlymixed and poured into petri dishes. The water was removed under vacuumat room temperature. After drying for approximately 36 hours, theresulting solid was ground using a mortar and pestle. 400 mg of theground keratin/albumin powder (320 mg keratin/80 mg albumin) was placedinto each of several two-part gelatin capsules (No. 0, Eli Lilly andCo., Indianapolis, Ind.). The dissolution profile of these tablets wasmeasured using the rotating paddle method.

[0105] When the albumin was mechanically mixed with absorbent keratin inpowder form (i.e. tablets), the disintegration and release were almostimmediate and complete in water. Release from the tablet in gastricfluid was also immediate, although the equilibrium amount of albumin wassignificantly lower, suggesting that some of the albumin remained boundto the keratin. This is presumably due to a pH effect on either thekeratin or the solubility or stability of the albumin in gastric fluid.An identical trend is evident in the data obtained from capsules.

[0106] Comparison of the tablet and capsule data reveals the effect ofthe different preparation procedures that were employed. In the case ofthe tablets, simple mechanical mixing resulted in more materialultimately being released, particularly in purified water, becausemechanical mixing does not result in a high degree of interaction at themolecular level. When intimate mixing was employed, as in the case ofthe gelatin capsules, much less material was released over an equivalenttime period. The intimate mixing was the result of first forming a gelfrom an albumin solution and keratin. In the hydrated state, albumin andkeratin molecules are more free to interact on a molecular level. Whilenot being bound to any theory, it is expected that intimate mixing wouldresult in more tightly bound albumin and consequently, less release thana system employing less intimate mixing. The unreleased material will beavailable however, and would be dissolved as the keratin degrades in abiological system.

[0107] 3. Phenylpropanolamine (Norephedrine) Preparations

[0108] Tablet samples that incorporated the vasoconstrictor norephedrinewere prepared by binding the drug to oxidized keratin in an ion exchangestep. Six different oxidized keratin samples were prepared by boiling 30g each of human hair in 500 mL of 2 w/v % H₂O₂ for 1, 2, 3, 4, 5 and 6hours. Longer oxidation times results in the generation of more sulfonicacid residues. More sulfonic acid residues results in increased bindingaffinity which in turn results in increased drug loading capabilitiesfor molecules that bind with sulfonic acid residues. Each of theoxidized keratin samples were ion exchanged with norephedrine. This wasaccomplished by dissolving a measured amount of norephedrine intoethanol, adding 5 g of oxidized keratin and heating to reflux for 2hours, followed by stirring at room temperature for 24 hours.

[0109] Equivalents of norephedrine bound to the keratin were determinedbased on the equivalents of sulfonic acid generated during the oxidationprocess. For example, 5 g of hair oxidized for 4 hours would beexchanged with approximately 5×10⁻³ moles of norephedrine. Thiscalculation is based on data generated for a pH 7 titration curve forH₂O₂ oxidized hair.

[0110] After binding the norephedrine in the ion exchange step, thesolid keratin was separated by filtration, dried, ground and pressedinto 500 mg tablets as described previously. The dissolution profile ofthese tablets was measured using the rotating paddle method.

[0111] These data show a general trend toward higher drug loading atlonger oxidation times, which is also suggested by the titration curve.As discussed previously, longer oxidation times results in increasedsulfonic acid residues, which in turn increases drug loading formolecules like norephedrine. In addition, longer oxidation timesresulted in longer sustained release as demonstrated by the 5 hour and 6hour oxidation samples. This is a kinetic effected based on the factthat longer oxidation times result in more sulfonic acid residues, morekeratin surface area after the solid has been ground prior to the ionexchange process, and more efficient ion exchange due to greaterdiffusion. All of these factors serve to increase binding in more highlyoxidized keratin.

[0112] Many drugs of interest are ionized at physiological pH. Thesedrugs exist as a cation, anion or zwitterion (both cation and anion).These drugs require a counterion (with the exception of a zwitterion)with an opposite charge of equal magnitude. Since proteins contain bothanionic (carboxylate, imidizole and, in the case of oxidized keratin,sulfonic acid) and cationic (amine and guanidyl) functionalities atphysiological pH, drugs capable of forming a complimentary couterion tothese functionalities will exhibit binding affinity.

[0113] The degree of binding may be estimated from the pKa of the drugof interest and the pH of the keratin protein in solution. The estimateis calculated by determining the pKa of the drug in its normalphysiological form. If there is a substantial difference (>1) in the pKaof the drug versus the pH of the keratin, there is a possibility ofbinding. For example, drugs that contain amine functionalities are inthe protonated form at physiological pH. The pKa of a protonated amineusually ranges from 9 to 10. Thus a protein that is acidic in aqueoussolution will readily protonate a drug containing an amine forming astable ammonium compound. Keratin absorbent produces a pH of 3.0 to 4 inaqueous solution. The binding in this case is ionic arising from theacid-base interaction of the protein and the drug. The number of bindingsites can be garnered from the number of acid equivalents in the proteinwhich in turn can be determined by titration.

[0114] 4. 4-Acetminophenol (Acetaminophen) Preparations

[0115] Samples that incorporated the antipyretic acetaminophen wereprepared by binding the drug to oxidized keratin in an ion exchangestep. Six different oxidized keratin samples were prepared by boiling 30g each of human hair in 500 mL of 2 w/v % H₂O₂ for 1, 2, 3, 4, 5 and 6hours. This was done in order to generate samples with different amountsof sulfonic acid residues, hence, different binding affinities andpresumably, different release characteristics. Compounds with weakability to form amine salts, such as acetaminophen, are expected to formacid-base interactions with the keratin molecules. Acetaminophenmolecules also contain a phenolic functionality, which can participatein binding through Van der Waal's interactions. These binding mechanismsare similar to the ion exchange process described for norephedrine, butresult in a less tightly bound drug. This binding was accomplished bydissolving a measured amount of acetaminophen into ethanol, adding 5 gof oxidized keratin and heating to reflux for 2 hours, followed bystirring at room temperature for 24 hours. Acetaminophen binding is mostlikely dominated by Van der Waal's interactions. pH is important only inso far as it effects the kinetics of the process. A sufficiently opentertiary structure for the keratin is important in the ionic exchangesimply to promote interactions between molecules. The openness of thekeratin is dependant on the oxidation so, indirectly, pH is important inVan der Waal's binding. Measured amounts of acetaminophen were estimatedbased on the amount of time the hair had been oxidized. The pH 7titration curve for H₂O₂ oxidized hair was used as a guide for theloading of keratin with acetaminophen.

[0116] After binding the acetaminophen in the ion exchange step, thesolid keratin was separated by filtration, dried, ground and pressedinto 500 mg tablets as described previously. The dissolution profile ofthese tablets was measured using the rotating paddle method.

[0117] Evaluation of the loading of acetaminophen was accomplished bydisintegrating a single tablet into a known volume of water over a oneweek period at room temperature. In addition, that samples weresonicated for 90 minutes prior to sampling. A sample of the solution waswithdrawn and analyzed by HPLC.

[0118] The curves do not suggest a clear release trend relative tokeratin oxidation time. This may be due to the fact that acetaminophenis a multifunctional molecule and can potentially bind through more thanone mechanism. However, these mechanisms do not appear to result intremendously strong interactions, or large numbers of interactionsbetween acetaminophen and keratin. This is suggested by the overall lowloading levels. The drug compound that does become bound, however, isreleased at a much slower rate than norephedrine (5 hours versus 2 hoursrespectively). This is particularly true for the 4-hour oxidationsample.

[0119] 5. Formulations

[0120] The following formulations are examples of the types offormulations in which a keratin excipient of the present invention maybe formulated. Formulations, however, are not limited to the followingnon-exclusive list and a keratin excipient of the present invention maybe formulated in any appropriate formulation which may be identified byone of ordinary skill in the art without undue experimentation.

[0121] (a) Liposomes

[0122] Techniques for producing unilamellar liposomes and multilamellarliopsomes are disclosed in U.S. Pat. Nos. 4,837,028, 4,522,803,4,501,728, 4,310,506, 4,235,871, 4,224,179, 4,078,052, 4,394,372,4,308,166, 4,485,054 and 4,508,703 (all herein incorporated byreference). Methods of production of liposomes are also reviewed inSzoka, et al., (1980). A keratin excipient of the present invention maybe incorporated in a liposomal preparation such that the preparationcomprises about 0.5, or about 1.0 or about 5.0, or about 10, or betweenabout 0.5 to about 5.0, or between about 1 to about 10 percent by weightof the keratin excipient.

[0123] (b) Water-in-Oil Emulsion

[0124] Following is an exemplary water-in-oil type emulsion formulation:A. Mixture of a higher molecular 20.00% fatty alcohol, wax esters andfats Decyl oleate 10.00% White petroleum jelly 10.00% Triglycerolmixture of various 10.00% natural fats p-Hydroxybenzoic acid propylester 0.02% B. Water 49.08% Dehydracetate 0.20% p-Hydroxybenzoic acidmethyl ester 0.20% C. Keratin excipient 1.00% D. Perfume oil 0.50%

[0125] The components mentioned under part A are mixed and warmed to 70°C. Mixture B is boiled up, cooled to 75° C. and added to part A, whilststirring. Whilst stirring, the mixture is cooled to 35° C. and, parts Cand D are added. Amounts given as percentage by weight.

[0126] (c) Oil-in-Water Type Emulsion

[0127] Following is an exemplary oil-in-water emulsion: A.Self-emulsifying mixture of monogly- 16.00% cerides and diglycerides ofhigher, saturated fatty acids Fatty alcohol polyglycol ether 1.00%2-Octyldodecanol 6.00% Isopropyl myristate 4.00% p-Hydroxybenzoic acidpropyl ester 0.02% B. Glycerol 6.00% Water 65.08% Na dehydracetate 0.02%p-Hydroxybenzoic acid methyl ester 0.02% C. Keratin excipient 1.00% D.Perfume oil 0.05%

[0128] Preparation similar to subsection 4.b supra. Amounts given aspercentage by weight.

[0129] (d) Cream Formulation

[0130] Following is an exemplary cream formulation: A. Sodiumcetylstearyl sulphate 0.8% Cetylstearyl alcohol 7.2% Wool grease 2.0%Isopropyl palmitate 14.5% 2-Octyl-dodecanol 10.0% B. Water(demineralised) 59.5% Propylene 1,2-glycol 5.0% C. Keratin excipient1.0% D. Perfume oil 9.5% Preservative 9.5%

[0131] Preparation similar to subsection 4.b supra. Amounts given aspercentage by weight.

[0132] (e) Lotion Formulation

[0133] Following is an exemplary lotion formulation: A. Decyl oleate2.5% Isopropyl myristate 2.5% Liquid paraffin 4.0% Polyoxyethylenestearate 0.9% Sorbitane monostearate 0.6% B. Water (demineralised) 80.4%Ethyl alcohol 10.0% Allantoin 0.1% C. Keratin excipient 1.0% D. Perfumeoil 8.5% Preservative 8.5%

[0134] Preparation similar to subsection 4.b supra. Amounts given aspercentage by weight.

[0135] The formulation of a keratin excipient of the present inventionis not limited to 1.0 percent by weight as shown in the exemplifiedemulsion, cream, or lotion formulations. The keratin excipient may beformulated at any appropriate percentage which may be, but not limitedto, about 0.5, or about 1.0, or about 5.0, or about 10.0, or betweenabout 0.5 to about 5.0, or between about 1.0 to about 10.0 percent byweight of the keratin excipient.

[0136] E. Cell Scaffold and Tissue Engineering Applications

[0137] The use of implantable organ or tissue equivalents has been thesubject of considerable research and development. One approach is toattach isolated cells onto biocompatable scaffolds in vitro and thenimplant the polymer-cell scaffold into recipients (Vacanti, 1988).Methods and examples of the preparation of scaffolds and their use toimplant diverse cell types are disclosed in U.S. Pat. Nos. 5,041,138,5,736,372, 5,759,830, 5,770,193, 5,770,417, 5,964,807, 5,916,557 and5,981,825, which are herein incorporated by reference. An alternative tothe growth of cells on a scaffold in vitro for subsequent implantation,is the use of scaffolds to provide matrices for the infiltration andgrowth of endogenous cells in vivo. Examples of such are disclosed inU.S. Pat. Nos. 4,772,287 and 4,904,260, which are herein incorporated byreference. Thus tissue engineering scaffolds can be used for tissuerepair, tissue reconstruction and wound healing, as disclosed forhyaluronan based scaffolds in U.S. Pat. No. 5,939,323, hereinincorporated by reference.

[0138] Various hydrogels have been used to form three-dimensionalscaffolds suitable for tissue engineering. These include hydrogels madefrom 2-hydroxyethyl methacrylate (Plant et al., 1995; Santin et al.,1996), agarose (Bellamkonda et al., 1995, Dillon et al., 1998; Yu etal., 1999), gelatin (Kang et al., 1999), alginates (de Chalain et al.,1999; Rowley et al., 1999), and polysaccharide augmentedglycosaminoglycan (Sechriest et al., 2000). In addition to the formationof a scaffold to guide growth, injection of cells in hydrogels have beenshown to be retained at the delivery site and promote formation of thedesired regenerated tissue (Atala et al., 1993; Cao et al. 1998).

[0139] Keratins supply suitable substrata for the growth of cells. Akeratin sheet has been disclosed to support the proliferation of humankeratinocytes, fibroblasts and microvascular endothelial cells (U.S.Pat. No. 5,932,552). In addition, it has been shown that akeratin-coated substrata was more adhesive to fibroblasts thancomparative collagen or glass substrata (Yamauchi et al., 1998). Akeratin implant material has been disclosed in U.S. Pat. No. 5,358,935,which is herein incorporated by reference. The keratin hydrogel of thepresent invention provides a suitable material for the construction ofcell scaffolds. For example, the absorbent keratin, which into ahydrogel upon the addition of water, can be used in the nonwoven filmembodiment of the present invention. The nonwoven film may beconstructed with synthetic polymer webs, the synthetic polymers of whichmay be resorbable. A number of synthetic polymers have been disclosed assuitable structural materials for tissue engineering scaffolds, such asin U.S. Pat. Nos. 5,399,665, 5,981,825, 5,981,825, and 6,022,828, whichare herein incorporated by reference. In other embodiments of thepresent invention, the absorbable keratin powder may be incorporatedinto synthetic polymer fibers or other three dimensional shapes.

[0140] The keratin hydrogel of the present invention may be used as avehicle for the injection of cells into a specific tissue location. Forsuch a vehicle, the absorbent keratin powder of the present inventionwould be hydrated water or a medium suitable to maintain cell viability.Such media are well known to those of ordinary skill in the art. Mediacan be added to the absorbent keratin powder to tailor the viscosity forinjection. This viscosity will vary on the gauge size of the needleemployed and can be readily ascertained without undue experimentation byone of skill in the art. The injectable keratin hydrogel can also bedelivered to a support structure, which may be a permeable supportstructure, as disclosed in U.S. Pat. No. 6,027,744, hereby incorporatedby reference. The injectable keratin hydrogel can also be used as an invivo scaffold to fill soft tissue structural defects and replace tissue.The keratin hydrogel of the present invention is non-toxic andbiocompatible and may be used as a vehicle for various cell types,including, but not limited to keratinocytes, fibroblasts, chondrocytes,hepatocytes, splenocytes, osteoblasts, neurocytes and endothelial cells.

[0141] F. Hydrogel Stability

[0142] The stability of keratin hydrogels at body temperature wasevaluated for drug delivery, injectable implant and tissue engineeringapplications. The testing matrix employed varied keratin absorbentprocessing and formulation parameters. Processing parameters that werevaried included oxidation time (1-6 hr.), type of oxidant used(peracetic acid or hydrogen peroxide), and grinding time, which effecteda change in the particle size. Formulation parameters that were variedincluded solids content of the gel (1:8, 1:6 and 1:4 solids to liquid).

[0143] 1. Preparation of Hydrogels

[0144] The hydrogels were prepared as follows. Human hair obtained froma barbershop was washed with VersaClean™ detergent and dried by vacuumfiltration. The hair was oxidized by boiling in 2 w/v % solutions ofoxidant (either peracetic acid or hydrogen peroxide) at a solids toliquid ratio of ca. 1:17 for 1, 2, 3, 4, 5 and 6 hours. The oxidizedhair was rinsed with deionized water, filtered and dried. The pH of thehair was titrated to ca. 7 by exposure to a known amount of sodiumhydroxide. The number of moles of sodium needed to neutralize theoxidized hair was determined from titration curves for peracetic acidand hydrogen peroxide treated hair. Titration was affected by boilingthe hair in a solution of ethanol with sodium hydroxide for a 2 hourperiod. After cooling, the solution was continually stirred for anadditional 22 hours. The solid keratin was separated by filtration,rinsed with ethanol and dried under vacuum.

[0145] Grinding was performed in a 1-quart ball mill using 1 cm diameterceramic grinding media. Grinding times of 20, 40 and 60 minutes wereused so as to generate smaller keratin particles with longer grindingtimes. This resulted in the generation of 36 absorbent keratin powdersamples (2 oxidants×6 oxidation times×3 grinding times=36 samples). Eachpowder sample was then used to make hydrogels using pH 7.4 phosphatebuffered saline solution (lot no. 099H6118, [120 mmol/L NaCl, 2.7 mmol/LKCl and 10 mmol/L phosphate buffer], Sigma Diagnostics, St. Louis, Mo.63178). The weight ratio of solids to liquid was varied at 1:8, 1:6 and1:4, resulting in the generation of 108 samples (2 oxidants×6 oxidationtimes×3 grinding times×3 gels=108 samples). The gels were mixed in 4dram vials using a vortex mixer, capped, sealed with Parafilm™ andplaced in a heated oven at 37.5° C.±2° C. for in vitro stabilitytesting. Samples that did not form viscous gels were immediately removedfrom the oven and discarded. Visual observations were conducted on adaily basis throughout the study. Gels that exhibited a decrease intheir original viscosity were removed from the study and deemed to nolonger be stable. These samples were retained frozen for later analysis.

[0146] 2. Stability and Viscosity Results

[0147] During the preparation of these gels, a variety of viscosities inthe resulting hydrogels were observed. In general, gels with highersolids content were more viscous than gels with lower solids content(1:4>1:6>1:8). In addition, shorter grinding times (i.e. larger particlesize) also resulted in higher viscosities (20 min.>40 min.>60 min.).

[0148] Formulations that were not stable for at least one day werecategorized as non gel-forming as indicated on the graphs. Most samplesthat were stable for greater than 80 days do not represent the terminusof the test, simply the cutoff point for data collection. To furtherexamine stability, the samples are further observed for greater than 80days until the samples are no longer stable or until some defined time.Samples which were stable at the last data collection time point areindicated with a data label on the graph.

[0149] In general, these data suggest that more stable gels weregenerated from peracetic acid oxidized hair than from hydrogen peroxideoxidized hair (11 gels with stability greater than 120 days forperacetic acid versus 2 for peroxide). Secondly, gels with higher solidscontent appear to be more stable. Many more 1:4 gels were stable forlong time periods than gels with a lower solids content. Lastly, thesedata do not show any direct correlation between gel stability andparticle size (i.e. grinding time).

[0150] In this study 19 gels exceeded 80 days of stability and 13 gelsexceeded 120 days of stability. To further test these samples, they arefurther subjected to elevated temperature testing. Primarily, they aregels made from hair that had been oxidized with peracetic acid for 3, 4or 5 hours and formulated as 1:4 gels.

[0151] These data establish that keratin hydrogels can be processed toremain stable at human body temperature for time periods ranging fromdays to in excess of four months. At the time of degradation, the gelbecomes hydrolyzed to lower molecular wight polypeptides, some in therange of 1000 daltons, and likely becomes bioresorbable. Those gels thatdo not degrade are likely to remain biostable, thus demonstrating arange of biodegradation and resorption possibilities with these gelformulations. In addition, the gels can be formulated to significantlyvary viscosity, ranging from very fluid at low solids concentrations tohighly viscous at high solids concentrations.

[0152] G. Applications of Hydratable Keratin

[0153] One use for the keratin powder and fiber is as a disposablediaper filler material. Disposable diapers typically have an absorbentinner layer that is often filled with a superabsorbent polymer andcellulosic material, often chemically derived from wood pulp. In oneapplication of the keratin material, a layer of the hydratable keratinis positioned in a disposable diaper near the skin but separated fromthe skin by a permeable layer. The hydratable keratin layer can serve toabsorb urine and water from the wearer. In some embodiments, thehydratable keratin includes a substantial fraction of soluble peptideshaving wound healing properties, as discussed in co-pending U.S. patentapplication Ser. No. 09/330,550, filed Jun. 11, 1999, entitled SOLUBLEKERATIN PEPTIDE, herein incorporated by reference. The water-solublepeptides are believed to be entrained in the keratin structure and ableto leach out when water is applied. In use, the keratin layer remainsdry until soaked with urine, at which point the soluble peptides candiffuse out of the keratin. The soluble peptides dissolved in the liquidpresent can thus come in contact with the skin. The wound healingproperties of the peptides are believed to be beneficial in treatingdiaper rash.

[0154] In another use, the hydratable keratin powder or fiber can beused as an ingredient in cosmetics. In one application, the keratinpowder is admixed with other cosmetic ingredients. The keratin power,when brought into contact with water from the other cosmetic ingredientsor from the skin of the wearer, forms a hydrogel that forms a protectivelayer over the skin and also retains moisture against the skin. Thekeratin powder, which has beneficial properties for skin, is thus heldagainst the skin, moisturizing the skin. In some embodiments, thekeratin powder includes soluble peptides that can diffuse out of thepowder with application of water. The soluble peptides are believed tobe non-immunogenic, mitogenic, biocompatible and have beneficial skinhealing properties. Cosmetics including the hydratable keratin powdercan aid in both moisturizing and healing skin. Keratin powder can alsobe used as an absorbent replacement to talc, the most popular cosmeticbase, to which many are allergic. The keratin powder or fibrous materialcan be used to promote healing of damaged skin. The keratin material canbe applied to skin afflictions such as diaper rash, bums, sunburns,cuts, abrasions, punctures, sores, bed sores, ulcers, diabetic ulcers,irritated skin, surgical incisions, skin graft donor sites, and wrinkledskin. In one method, the keratin material is admixed with a carrier suchas a cream, lotion, or gel.

[0155] Other applications of the keratin solid include using the keratinpowder or fibers in feminine hygiene products, where the keratin canserve a moisture absorbing function. Yet another application is inanti-perspirants, where the keratin solid can absorb moisture. Stillanother application is in drug release applications, where the keratincan be used in powder, fiber, or film form to provide a moist, benignenvironment against the skin for drug release. The present invention, inpowder, fiber, and nonwoven sheet forms, is also believed suitable foruse in forming tissue-engineering scaffolds. An additional use as a foodadditive is contemplated, as some naturally-derived products, such asgelatin, are already used in food products.

[0156] The keratin can also be used as the precursor to the formation ofa gel, which can form a keratin hydrogel upon the addition of water tothe absorbent keratin solid. The keratin can be used to form an in situgel. In the in situ application, the keratin powder can reside within anenvelope predisposed at a site and the water added into the envelopalready in position. The keratin can be stored in solid form, forexample as a fiber, powder, or some combination thereof, and water addedlater. Keeping the keratin in solid form allows for storage and latergel formation only when desired, as in an emergency medical fielddressing application. Requiring the keratin to pass through a solid stepalso serves to purify the resulting gel, as many impurities are removedin the intermediate processing steps.

[0157] The present invention can also be used to augment soft tissue.Keratin hydrogel precursor in powder form may be suspended in aninjectable carrier and injected subdermally. In one method, the keratinpowder is suspended in saline and injected subdermally.

[0158] The resulting hydrogel has been observed to have viscoelasticproperties, favorable for use as an implant filler such as a breastimplant. The hydrogel has been observed to flow more readily whenmanipulated, which may prove beneficial to implant applications wherethe consistency of the implant is important.

[0159] The present invention can be used in a wound dressing. One use ofhydratable keratin in a wound dressing is wound exudate management. Thushydratable keratin may be used as a component of an absorbent wounddressing. In one embodiment, the hydratable keratin may be used in theform of a nonwoven film. In another embodiment, hydratable keratin maybe used as a woven composition. In a woven composition, fibers ofhydratable keratin may be woven with natural fibers or synthetic polymerfibers. In another embodiment, hydratable keratin is enclosed by awater-permeable material which, while allowing passage of wound exudateand blood, prevents passage of the hydratable keratin solid of thepresent invention. Wound dressings include, but are not limited to,adhesive bandages and tapes. Skin-contact adhesive bandages and medicaltapes are well known to those of skill in the art. Adhesive bandagestypically comprise an absorbent pad, a backing and a pressure sensitiveadhesive to maintain the dressing in place. In one aspect of the presentinvention, an absorbent wound dressing comprising hydratable keratin isan absorbent pad of an adhesive bandage. Components, configurations anddelivery systems for adhesive bandages are disclosed in U.S. Pat. Nos.6,018,092, 5,947,917, 5,633,070, and 5,503,908, which are hereinincorporated by reference. Upon contact with moisture, which maybe inthe form of wound exudate or blood, the keratin fibers can form ahydrogel and leach water soluble peptides into the wound.

[0160] Numerous advantages of the invention covered by this documenthave been set forth in the foregoing description. It will be understood,however, that this disclosure is, in many respects, only illustrative.Changes may, be made in details, particularly in matters of reagents,concentrations, and step order, without exceeding the scope of theinvention. The invention's scope is, of course, defined in the languagein which the appended claims are expressed.

REFERENCES

[0161] The following references, to the extent that they provideexemplary procedural or other details supplementary to those set forthherein, are specifically incorporated by reference.

[0162] Agrawal, J. Biomed. Res. App. Biomaterials, 38: 105-14 (1997).

[0163] Atala et al., J. Urol. 150:745-47 (1993).

[0164] Bellamkonda et al., J. Biomed. Mater Res., 29:663-71 (1995).

[0165] Cao et al., Plast. Reconstr. Surg., 102:2293-98 (1998).

[0166] De Chalain et al., J. Biomed. Mater Res, 44:280-288 (1999).

[0167] Dillon et al., J. Biomatter. Sci. Polymer Ed., 9:1049-69 (1990).

[0168] Duranti et al., Dermatol. Sugery, 24:1317-25 (1998).

[0169] Flory, Principles of Polymer Chemistry, pp. 497-539, CornellUniversity Press, Ithaca and London (1953)

[0170] Franz, Curr. Probl. Dermatol., 7:309, 1978.

[0171] Kang et al., Biomaterials, 20: 1339-44 (1999).

[0172] Lee et al., Drug Dev. Indust. Pharm., 12:349, 1986.

[0173] Manna et al., J. Eur. Acad. Dermatol. Venereol, 13:183-92 (1999).

[0174] Plant et al., Brain Res. 6:119-130 (1995).

[0175] Rowley et al., Biomaterials, 20:45-53 (1999)

[0176] Santin et al., Biomaterials, 17:1459-67 (1996).

[0177] Sechriest et al., J. Biomed. Matter Res., 49:534-41 (2000).

[0178] Szoka, et al., Ann. Rev. Biophys. Bioeng., 9:465-508, 1980.

[0179] Vacanti, Archives of Surgery, 123: 545-549 (1988).

[0180] Yamauchi et al., J. Biomater Sci Polym Ed, 9:259-70, 1998

[0181] Yu et al., Tissue Eng. 5:291-304 (1999).

What is claimed is:
 1. A preparation comprising a cross-linked insolubleoxidized keratin excipient, wherein said cross-linked insoluble oxidizedkeratin is chemically modified to contain sulfonate groups.
 2. Thepreparation of claim 1, wherein said keratin is derived from hair, fur,nails, feet, beaks, feathers, horns or hooves.
 3. The preparation ofclaim 1, wherein said keratin is derived from hair.
 4. The preparationof claim 1, wherein said hair is human hair.
 5. The preparation of claim1, wherein said sulfonate groups are ionically associated with cations.6. The preparation of claim 5, wherein said cations comprise monovalentcations.
 7. The preparation of claim 6, wherein said monovalent cationscomprise sodium or potassium.
 8. The preparation of claim 1, whereinsaid preparation comprising a cross-linked insoluble oxidized keratinexcipient is prepared in a form selected from the group consisting of apowder, tablet, film, capsule, lotion, cream, gel, solution, suspension,emulsion and aerosol.
 9. The preparation of claim 1, further comprisingat least one additive selected from the group consisting of diluents,fillers, lubricants, stabilizers, binders and gelants.
 10. Thepreparation of claim 1, wherein said cross-linked insoluble oxidizedkeratin excipient comprises a hydratable cross-linked insoluble oxidizedkeratin.
 11. The preparation of claim 1, wherein said cross-linkedinsoluble oxidized keratin excipient is a cross-linked insolubleoxidized keratin hydrogel.
 12. The preparation of claim 1, furthercomprising an active agent.
 13. The preparation of claim 12, whereinsaid sulfonate groups are ionically associated with cations.
 14. Thepreparation of claim 13, wherein said cations comprise the cationic formof an active agent.
 15. The preparation of claim 12, wherein said activeagent is the free base of an active agent that may be otherwiseformulated as a hydrochloride.
 16. The preparation of claim 12, whereinsaid active agent is a pharmaceutical agent
 17. The preparation of claim16, wherein said pharmaceutical agent is a protein, polypeptide orpeptide.
 18. The preparation of claim 12, wherein said active agent is acosmetic agent.
 19. The preparation of claim 18, wherein said cosmeticingredient is a water soluble peptide derived from keratin.
 20. Thepreparation of claim 12, wherein said preparation comprising across-linked insoluble oxidized keratin excipient and an active agent isprepared in a form selected from the group consisting of a powder,tablet, film, capsule, lotion, cream, gel, solution, suspension,emulsion and aerosol.
 21. The preparation of claim 12, wherein saidpreparation comprising a cross-linked insoluble oxidized keratinexcipient is prepared in the form of a non-woven film.
 22. Thepreparation of claim 12, further comprising at least one additiveselected from the group consisting of diluents, fillers, lubricants,stabilizers, binders and gelants.
 23. The preparation of claim 12,wherein association of said active agent with said cross-linkedinsoluble oxidized keratin excipient provides for the controlled releaseof said active agent.
 24. The method of claim 23, wherein the ionicassociation of said active agent with said keratin excipient providesfor said controlled release of said active agent.
 25. The preparation ofclaim 23, wherein the hydrolysis of said keratin excipient provides forsaid controlled release of said active agent.
 26. The preparation ofclaim 12, wherein said cross-linked insoluble oxidized keratin excipientcomprises a cross-linked insoluble oxidized hydratable keratin.
 27. Thepreparation of claim 12, wherein said cross-linked insoluble oxidizedkeratin excipient is a cross-linked insoluble oxidized keratin hydrogel.28. An absorbent wound dressing comprising the cross-linked insolubleoxidized keratin excipient of claim
 1. 29. The absorbent wound dressingof claim 28, wherein said absorbent wound dressing is the absorbent padof an adhesive bandage.
 30. The absorbent wound dressing of claim 28,further comprising an active agent.
 31. A drug delivery devicecomprising the cross-linked insoluble oxidized keratin excipient ofclaim
 12. 32. The device of claim 31, wherein said device is atransdermal drug delivery device.
 33. The device of claim 31, whereinsaid device is an inhalation drug delivery device.
 34. The device ofclaim 31, wherein said device is an oral drug delivery device.
 35. Thedevice of claim 31, wherein said device is an implantable drug deliverydevice.
 36. A preparation comprising a keratin excipient associated withan active agent, wherein said keratin excipient is chemically modifiedto contain sulfonate groups.
 37. The preparation of claim 36, whereinsaid keratin is derived from hair, fur, nails, feet, beaks, feathers,horns or hooves.
 38. The preparation of claim 36, wherein said keratinis derived from hair.
 39. The preparation of claim 36, wherein said hairis human hair.
 40. The preparation of claim 1, wherein said keratinexcipient is a cross-linked insoluble oxidized keratin excipient. 41.The preparation of claim 36, wherein said sulfonate groups are ionicallyassociated with cations.
 42. The preparation of claim 41, wherein saidcations comprise the cationic form of an active agent.
 43. Thepreparation of claim 42, wherein said cations comprise monovalentcations.
 44. The preparation of claim 43, wherein said monovalentcations comprise sodium or potassium.
 45. The preparation of claim 36,wherein said active agent is the free base of an active agent that maybe otherwise formulated as a hydrochloride.
 46. The preparation of claim36, wherein said active agent is a pharmaceutical agent
 47. Thepreparation of claim 46, wherein said pharmaceutical agent is a protein,polypeptide or peptide.
 48. The preparation of claim 36, wherein saidactive agent is a cosmetic agent.
 49. The preparation of claim 48,wherein said cosmetic ingredient is a water soluble peptide derived fromkeratin.
 50. The preparation of claim 36, wherein said preparationcomprising a keratin excipient is prepared in a form selected from thegroup consisting of a powder, tablet, film, capsule, lotion, cream, gel,solution, suspension, emulsion and aerosol.
 51. The preparation of claim36, wherein said preparation comprising a keratin excipient is preparedin the form of a non-woven film.
 52. The preparation of claim 36,further comprising at least one additive selected from the groupconsisting of diluents, fillers, lubricants, stabilizers, binders andgelants.
 53. The preparation of claim 36, wherein association of saidactive agent with said keratin excipient provides for the controlledrelease of said active agent.
 54. The method of claim 53, wherein theionic association of said active agent with said keratin excipientprovides for the controlled release of said active agent.
 55. Thepreparation of claim 53, wherein the hydrolysis of said keratinexcipient provides for the controlled release of said active agent. 56.The preparation of claim 36, wherein said keratin excipient comprises ahydratable keratin.
 57. The preparation of claim 36, wherein saidkeratin excipient is a keratin hydrogel
 58. An absorbent wound dressingcomprising the keratin excipient of claim
 36. 59. The absorbent wounddressing of claim 58, wherein said absorbent wound dressing is theabsorbent pad of an adhesive bandage.
 60. A drug delivery devicecomprising the keratin excipient of claim
 36. 61. The device of claim60, wherein said device is a transdermal drug delivery device.
 62. Thedevice of claim 60, wherein said device is an inhalation drug deliverydevice.
 63. The device of claim 60, wherein said device is an oral drugdelivery device.
 64. The device of claim 60, wherein said device is animplantable drug delivery device.
 65. A preparation comprising across-linked insoluble oxidized keratin excipient containing sulfonategroups, wherein an active agent is ionically associated with saidsulfonate groups.
 66. The preparation of claim 65, wherein said keratinis derived from hair, fur, nails, feet, beaks, feathers, horns orhooves.
 67. The preparation of claim 65, wherein said keratin is derivedfrom hair.
 68. The preparation of claim 65, wherein said hair is humanhair.
 69. The preparation of claim 65, wherein said active agent is acation.
 70. The preparation of claim 69, wherein said cation is apharmaceutical agent.
 71. The preparation of claim 69, wherein saidcation is a cosmetic agent.
 72. The preparation of claim 65, whereinionic association of said active agent with said cross-linked insolubleoxidized keratin excipient provides for the controlled release of saidactive agent.
 73. The preparation of claim 65, wherein the hydrolysis ofsaid cross-linked insoluble oxidized keratin excipient provides for thecontrolled release of said active agent.
 74. The preparation of claim65, wherein said preparation comprising a cross-linked insolubleoxidized keratin excipient is prepared in a form selected from the groupconsisting of a powder, tablet, film, capsule, lotion, cream, gel,solution, suspension, emulsion and aerosol.
 75. The preparation of claim65, wherein said preparation comprising a cross-linked insolubleoxidized keratin excipient is prepared in the form of a non-woven film.76. The preparation of claim 65, further comprising at least oneadditive selected from the group consisting of diluents, fillers,lubricants, stabilizers, binders and gelants.
 77. An absorbent wounddressing comprising the cross-linked insoluble oxidized keratinexcipient of claim
 65. 78. The absorbent wound dressing of claim 43,wherein said absorbent wound dressing is the absorbent pad of anadhesive bandage.
 79. A method for the delivery of an active agentcomprising providing an active agent in a keratin excipient, whereinsaid keratin excipient contains sulfonate groups.
 80. The method ofclaim 79, wherein said keratin excipient is a cross-linked insolubleoxidized keratin excipient.
 81. The method of claim 79, wherein saidactive agent is a pharmaceutical agent or a cosmetic agent.
 82. Themethod of claim 79, wherein said keratin is obtained from hair, fur,nails, feet, beaks, feathers, horns or hooves.
 83. The method of claim79, wherein said keratin is obtained from hair.
 84. The method of claim83, wherein said hair is human hair.
 85. The method of claim 79, whereinsaid active agent is ionically associated with said sulfonate groups.86. The method of claim 79, wherein said keratin excipient is deliveredto the lungs of an animal by inhalation of particles of said keratinexcipient.
 87. The method of claim 86, wherein said keratin excipientparticles are less than about 1 micron in diameter.
 88. The method ofclaim 79, wherein said delivery is transdermal delivery.
 89. The methodof claim 79, wherein said delivery is oral delivery.
 90. The method ofclaim 79, wherein said keratin excipient is implanted.
 91. The method ofclaim 79, wherein said delivery of said active agent provides for thecontrolled delivery of said active agent.
 92. The method of claim 79,wherein the ionic association of said active agent with said keratinexcipient provides for the controlled release of said active agent. 93.The method of claim 79, wherein the hydrolysis of said keratin excipientprovides for the controlled release of said active agent.
 94. The methodof claim 79, wherein the ionic association of said active agent withsaid cross-linked insoluble oxidized keratin excipient and thehydrolysis of said cross-linked insoluble oxidized keratin excipientprovide for the controlled release of said active agent.
 95. A processfor the preparation of a preparation comprising an active agent bound toa keratin excipient, wherein said active agent is ionically associatedwith said sulfonate group, comprising the steps: (a) oxidizing keratinin a first solution comprising a soluble oxidizing agent, such that aportion of the disulfide bonds of said keratin are oxidized to formsulfonic acid residues to obtain an oxidized solid fraction; (b)separating said oxidized solid fraction from said first solution;contacting said oxidized solid fraction with a second solutioncomprising a cationic form of an active agent dissolved in a solvent;(c) maintaining said second solution containing said oxidized solidfraction and said active agent for a time and at a temperature effectiveto cause an ionic interaction between said sulfonic acid residues andsaid cationic form of said active agent; and (d) substantially removingthe solvent from said salt solution to obtain a solid preparation. 96.The process of claim 95, further comprising grinding said solidpreparation to form a powder.
 97. The process of claim 95, wherein saidactive agent is a pharmaceutical agent or a cosmetic agent capable offorming a cation
 98. The process of claim 95, wherein said keratin isobtained from hair, fur, nails, feet, beaks, feathers, horns or hooves.99. The process of claim 95, wherein said keratin is obtained from hair.100. The process of claim 99, wherein said hair is human hair.
 101. Apreparation made by the process of claim
 95. 102. A process for thepreparation of a hydratable keratin excipient comprising the steps: (a)oxidizing keratin in a first solution comprising a soluble oxidizingagent, such that a portion of the disulfide bonds of said keratin areoxidized to form sulfonic acid residues to obtain an oxidized solidfraction; (b) separating said oxidized solid fraction from said firstsolution; contacting said oxidized solid fraction with a second solutioncomprising monovalent cations dissolved in a solvent; (c) maintainingsaid second solution containing said oxidized solid fraction and saidmonovalent cations for a time and at a temperature effective to cause aninteraction between said sulfonic acid residues and said monovalentcations to obtain a salt solution of said keratin and said monovalentcations; and (d) substantially removing the solvent from said saltsolution to obtain a hydratable keratin solid.
 103. The process of claim102, further comprising grinding said hydratable keratin solid to form apowder.
 104. The process of claim 102, further comprising contactingsaid hydratable keratin with an aqueous solution to form a hydrogel.105. The process of claim 104, further comprising adding an active agentto said aqueous solution before contacting said hydratable keratin toform said hydrogel.
 106. The process of claim 104, further comprisingadmixing an active agent with said hydratable keratin solid.
 107. Theprocess of claim 106, further comprising contacting said admixture ofsaid hydratable keratin and said active agent with an aqueous solutionto form a hydrogel.
 108. The process of claim 104, wherein said keratinis obtained from hair, fur, nails, feet, beaks, feathers, horns orhooves.
 109. The process of claim 104, wherein said keratin is obtainedfrom hair.
 110. The process of claim 109, wherein said hair is humanhair.
 111. A preparation made by the process of claim
 105. 112. Apreparation made by the process of claim
 106. 113. A process for thepreparation of a preparation comprising a cross-linked insolubleoxidized keratin excipient comprising the steps: (a) oxidizing keratinin a first solution comprising a soluble oxidizing agent, such that aportion of the disulfide bonds of said keratin are oxidized to formsulfonic acid residues to obtain an oxidized cross-linked insolublesolid fraction; (b) separating said oxidized cross-linked insolublesolid fraction from said first solution; and (c) substantially removingthe solvent from said oxidized cross-linked insoluble solid fraction.114. The process of claim 113, further comprising grinding said oxidizedcross-linked insoluble solid fraction to form a powder.
 115. The processof claim 113, wherein said keratin is obtained from hair, fur, nails,feet, beaks, feathers, horns or hooves.
 116. The process of claim 113,wherein said keratin is obtained from hair.
 117. The process of claim116, wherein said hair is human hair.
 118. A preparation made by theprocess of claim
 113. 119. A liposome preparation comprising a keratinexcipient associated with an active agent, wherein said keratin ischemically modified to contain sulfonate groups.
 120. The preparation ofclaim 119, wherein said keratin is derived from hair, fur, nails, feet,beaks, feathers, horns or hooves.
 121. The preparation of claim 119,wherein said keratin is derived from hair.
 122. The preparation of claim121, wherein said hair is human hair.
 123. The preparation of claim 119,wherein said sulfonate groups are ionically associated with cations.124. The preparation of claim 123, wherein said cations comprise thecationic form of an active agent.
 125. The preparation of claim 123wherein said cations comprise monovalent cations.
 126. The preparationof claim 125, wherein said monovalent cations comprise sodium orpotassium.
 127. The preparation of claim 119, wherein said active agentis the free base of an active agent that may be otherwise formulated asa hydrochloride.
 128. The preparation of claim 119, wherein said activeagent is a pharmaceutical agent
 129. The preparation of claim 128,wherein said pharmaceutical agent is a protein, polypeptide or peptide.130. The preparation of claim 119, wherein said active agent is acosmetic agent.
 131. The preparation of claim 130, wherein said cosmeticingredient is a water soluble peptide derived from keratin.
 132. Thepreparation of claim 119, wherein association of said active agent withsaid keratin excipient provides for the controlled release of saidactive agent.
 133. The preparation of claim 119, wherein said keratinexcipient comprises a hydratable keratin.
 134. The preparation of claim119, wherein said keratin excipient is a keratin hydrogel.